Immunological biomarker for predicting clinical effect of cancer

ABSTRACT

The present invention relates to the prediction of responsiveness to cancer immunotherapy of a subject based on the T-cell composition of the subject, and a therapeutic method using cancer immunotherapy based on the prediction. The present invention also provides a method for improving or maintaining responsiveness to cancer immunotherapy of a subject. Responsiveness to cancer immunotherapy is predict by determining a relative value of a CD4+ T-cell subpopulation, dendritic cell subpopulation, and/or CD8+ T-cell subpopulation correlated with a dendritic cell stimulation in an anti-tumor immune response in a sample derived from a subject. A composition for treating or preventing cancer comprising cells such as CD62LlowCD4+ T-cells is also provided.

TECHNICAL FIELD

The present invention relates to the field of cancer immunotherapy. Morespecifically, the present invention relates to the prediction ofresponsiveness to cancer immunotherapy of a subject based on the T-cellcomposition of the subject, and a therapeutic method using cancerimmunotherapy based on the prediction. In another aspect, the presentinvention provides a method of improving or maintaining responsivenessto cancer immunotherapy of a subject.

BACKGROUND ART

Cancer immunotherapy has drawn attention in recent years as having fewerside effects and exhibiting a greater effect compared to conventionalanticancer therapies targeting the metabolism of cancer cells or thelike (alkylating agents, platinum formulations, antimetabolites,topoisomerase inhibitors, microtubule polymerization inhibitors,microtubule depolymerization inhibitors, and the like). Among cancerimmunotherapies, anti-PD-1 immune checkpoint inhibition has drawnparticularly significant interest.

An anti-PD-1 antibody nivolumab is superior to docetaxel, which wasconventionally the standard therapy as a secondary therapy of non-smallcell lung cancer, by a large margin in all survival periods, thusbecoming the standard therapy with a recommendation level of A in theLung Cancer Society Guidelines (Brahmer J, et al. N Engl J Med 2015;373: 123-135). Pembrolizumab, which is also an anti-PD-1 antibody, issuperior to cytotoxic anticancer agents, which were conventionally thestandard therapy in primary therapy, in all survival periods (note: inpatients with expression of PD-L1 on tumor cells at 50% or greater). Ithas been decided that this will be the standard therapy for non-smallcell lung cancer in the future.

The effect of anti-PD-1 antibodies is not limited to lung cancer. Theeffect is about to be proven in renal cancer, head and neck cancer,gastrointestinal cancer, gynecological cancer, malignant lymphoma, andbreast cancer. Renal cancer is covered under insurance in Japan. Nextyear, head and neck cancer, gastrointestinal cancer, and malignantlymphoma are expected to be covered.

While anti-PD-1 antibodies appear to have achieved significant clinicalsuccess, anti-PD-1 antibodies in fact have significant problems.“Ineffective group”, whose condition worsens within three months inalmost all anti-PD-1 antibody clinical trials, is found from data forprogression free survival (PFS). Meanwhile, in groups for whichanti-PD-1 antibodies were effective for 1 year or longer, exacerbationin conditions was hardly observed thereafter, thus revealing that astate close to being healed is attained. This suggests the presence ofthree different subgroups, i.e., “ineffective group”, “highly effectivegroup” and “intermediate group” in terms of clinical effects, but abiomarker for the prediction thereof is not known. Administration ofanti-PD-1 antibodies, which are expected to be the standard therapy inalmost all cancer and tumor, to ineffective groups accounting for about40%, which would be not only a medical problem, but also a problem formedical economics.

CITATION LIST Non Patent Literature

-   [NPL 1] Brahmer J, et al. N Engl J Med 2015; 373: 123-135

SUMMARY OF INVENTION Solution to Problem

The present invention provides a method of using the composition of CD4⁺T-cells of a subject as an indicator for predicting a response to cancerimmunotherapy of the subject. The present invention also provides amethod of using the composition of dendritic cells and/or CD8⁺ T-cellsof a subject as an indicator for predicting a response of the subject toimmunotherapy. The present invention is partially based on the inventorsdiscovering that the responsiveness to cancer immunotherapy isassociated with the composition of T-cells and/or dendritic cells in asubject, and the responsiveness can be used as a biomarker. Thebiomarker of the present invention has a much higher level ofsensitivity and specificity than conventionally studied biomarkers.

The inventors have discovered that the three groups of therapeuticeffects to cancer immunotherapy (e.g., anti-PD-1 therapy or anti-PD-L1therapy), i.e., progressive disease (PD), stable disease (SD), andresponse (complete response (CR)+partial response (PR)), each exhibitsdifferent immunological conditions. Some of the embodiments of thepresent invention provide a method of predicting a response to cancerimmunotherapy as either progressive disease (PD), stable disease (SD),or response (complete response (CR)+partial response (PR)) when cancerimmunotherapy is applied to a subject. In the present invention, itshould be noted that a population of subjects which includes completeresponse group (CR) with a partial response group (PR), or a populationof subjects which includes a complete response group (CR) without apartial response group (PR), can be identified to be the same as apartial response group (PR).

One embodiment of the present invention is a method of using a relativeamount of a CD4⁺ T-cell subpopulation correlated with a dendritic cellstimulation in an anti-tumor immune response as an indicator forpredicting a response to cancer immunotherapy of a subject. Examples ofCD4⁺ T-cell subpopulations correlated with a dendritic cell stimulationin an anti-tumor immune response include, but are not limited to, CD4⁺T-cell subpopulations with decreased expression of a homing molecule toa secondary lymphoid organ, CD4⁺ T-cell subpopulations primed by aneffector T-cell, CD4⁺ T-cell subpopulations and regulatory T-cellsubpopulations primed by antigen recognition. The relative amount of aCD4⁺ T-cell subpopulation is selected from the group consisting of:

a ratio of a CD62L^(low)CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CCR7⁻CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD45RA⁻CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD45RO⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a LAG-3⁺CD62L^(low)CD4⁺ T-cell subpopulation inCD62L^(low)CD4⁺ T-cells;a ratio of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation in CD62L^(low)CD4⁺ T-cells;a ratio of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CCR4⁺CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD127⁺CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells; anda ratio of a Foxp3⁺CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;but is not limited thereto. The present invention provides, for example,a method of using a ratio of CD62L^(low) T-cells in CD4⁺ T-cells of asubject as an indicator for predicting a response to cancerimmunotherapy of a subject. In one embodiment, the method comprisesdetermining the ratio of CD62L^(low) T-cells in CD4⁺ T-cells in a samplederived from a subject. The ratio being higher than a threshold value(ineffective group threshold value) can indicate that the subject is nota part of an ineffective group with respect to the cancer immunotherapy.

Another embodiment of the present invention is a method of using arelative amount of a dendritic cell subpopulation correlated with adendritic cell stimulation in an anti-tumor immune response as anindicator for predicting a response to cancer immunotherapy of asubject. Examples of dendritic cell subpopulations correlated with adendritic cell stimulation in an anti-tumor immune response include, butare not limited to, dendritic cell subpopulations that increase due toan increase in a cell subpopulation with decreased expression of ahoming molecule in a CD4⁺ T-cell population and dendritic cellsubpopulations that increase due to an increase in a CD4⁺ T-cellsubpopulation primed by an effector T-cell in a CD4⁺ T-cell population.Examples of dendritic cell subpopulations include, but are not limitedto, HLA-DR⁺ dendritic cell subpopulations, CD80⁺ dendritic cellsubpopulations, CD86⁺ dendritic cell subpopulations, and PD-L1⁺dendritic cell subpopulations. Examples of dendritic cells include, butare not limited to, myeloid dendritic cells (mDC, CD141⁺CD11c⁺ dendriticcells) and plasmacytoid dendritic cells (pDC, CD123⁺CD11c⁺ dendriticcells).

Another embodiment of the present invention is a method of using arelative amount of a CD8⁺ T-cell subpopulation correlated with adendritic cell stimulation in an anti-tumor immune response as anindicator for predicting a response to cancer immunotherapy of asubject. Examples of CD8⁺ T-cell subpopulation correlated with adendritic cell stimulation in an anti-tumor immune response include, butare not limited to, CD8⁺ T-cell subpopulations that increase due to anincrease in a cell subpopulation with decreased expression of a homingmolecule in a CD4⁺ T-cell population, CD8⁺ T-cell subpopulations thatincrease due to an increase in a CD4⁺ T-cell subpopulation primed by aneffector T-cell in a CD4⁺ T-cell population, CD8⁺ T-cell subpopulationsthat increase due to an increase in a CD4⁺ T-cell subpopulation primedby antigen recognition in a CD4⁺ T-cell population, CD8⁺ T-cellsubpopulations that increase due to an increase in a HLA-DR+ dendriticcell subpopulation in a dendritic cell population, CD8⁺ T-cellsubpopulations that increase due to an increase in a CD80⁺ T-cellsubpopulation in a dendritic cell population, and CD8⁺ T-cellsubpopulations that increase due to an increase in a PD-L1⁺ dendriticcell subpopulation in a dendritic cell population. Furthermore, examplesof CD8⁺ T-cell subpopulations correlated with a dendritic cellstimulation in an anti-tumor immune response include, but are notlimited to, CD62L^(low)CD8⁺ T-cell subpopulation, CD137⁺CD8⁺ T-cellsubpopulation, and CD28⁺CD62L^(low)CD8⁺ T-cell subpopulation.

One embodiment of the present invention is a method of using an amountselected from: an amount of a CD4⁺ T-cell subpopulation correlated witha dendritic cell stimulation in an anti-tumor immune response;

an amount of a dendritic cell subpopulation correlated with a dendriticcell stimulation by a CD4⁺ T-cell in an anti-tumor immune response;an amount of a CD8⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;an amount of regulatory T-cells or a CD4⁺ T-cell subpopulationcorrelated with regulatory T-cells; andan amount of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation;in a subject as a variable (indicator) of a formula for predicting aresponse to cancer immunotherapy of the subject. In one embodiment,variables (X, Y) of the present invention are each selected from thegroup consisting of:an amount of a CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a CCR7⁻CD4⁺ T-cell subpopulation;an amount of a LAG-3⁺CD62L^(low)CD4⁺ T-cell subpopulation;an amount of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a CD45RA⁻CD4⁺ T-cell subpopulation;an amount of a CD45RO⁺CD4⁺ T-cell subpopulation;an amount of a CCR4⁺CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD127⁺CD25⁺CD4⁺ T-cell subpopulation; an amount of aCD45RA⁻ Foxp3⁺CD4⁺ T-cell subpopulation;an amount of a Foxp3⁺CD25⁺CD4⁺ T-cell subpopulation;an amount of an HLA-DR⁺ dendritic cell subpopulation;an amount of a CD80⁺ dendritic cell subpopulation;an amount of a CD86⁺ dendritic cell subpopulation;an amount of a PD-L1⁺ dendritic cell subpopulation;an amount of a CD62L^(low)CD8⁺ T-cell subpopulation;an amount of a CD137⁺CD8⁺ T-cell subpopulation; andan amount of a CD28⁺CD62L^(low)CD8⁺ T-cell subpopulation.

In the present invention, (X) can be, for example, a value selected fromthe group consisting of:

an amount of a CD4⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;an amount of a dendritic cell subpopulation correlated with a dendriticcell stimulation by a CD4⁺ T-cell in an anti-tumor immune response; andan amount of a CD8⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response. The method of thepresent invention can also calculate variables (X, Y), with a valueselected from the group consisting of:an amount of a CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a CCR7⁻CD4⁺ T-cell subpopulation;an amount of an LAG-3⁺CD62L^(low)CD4⁺ T-cell subpopulation;an amount of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a CD45RA⁻CD4⁺ T-cell subpopulation;an amount of a CD45RO⁺CD4⁺ T-cell subpopulation;an amount of an HLA-DR⁺ dendritic cell subpopulation;an amount of a CD80⁺ dendritic cell subpopulation;an amount of a CD86⁺ dendritic cell subpopulation;an amount of a PD-L1⁺ dendritic cell subpopulation;an amount of a CD62L^(low)CD8⁺ T-cell subpopulation;an amount of a CD137⁺CD8⁺ T-cell subpopulation; andan amount of a CD28⁺CD62L^(low)CD8⁺ T-cell subpopulation;

as (X).

For example, the method of the present invention can calculate variables(X, Y), with the amount of a regulatory T-cell subpopulation or theamount of a CD4⁺ T-cell subpopulation correlated with a regulatoryT-cell as (Y). The method of the present invention can also calculatevariables (X, Y), a value selected from the group consisting of:

an amount of a CCR4⁺CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD127⁺CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation; andan amount of a CD4⁺Foxp3⁺CD25⁺ T-cell subpopulation;

as (Y).

The method of the present invention can use, for example, a comparisonof a relative value of X to Y with a threshold value (ineffective groupthreshold value) comprising measuring the amount of CD4⁺CD62L^(low)T-cells (X) and measuring the amount of CD4⁺Foxp3⁺CD25⁺ T-cells (Y) asindicators for predicting that the subject is not a part of anineffective group with respect to the cancer immunotherapy. The amountof a regulatory T-cell subpopulation or the amount or ratio of a CD4⁺T-cell subpopulation correlated with a regulatory T-cell can be used as(Y). In particular, examination of the ratio of CD62L^(low)CD4⁺T-cells/regulatory T-cells as a biomarker have not been reported up tothis point, but the inventors have discovered that this ratio is veryuseful as a biomarker for predicting responsiveness to cancerimmunotherapy.

The method of the present invention can use, for example, a comparisonof a relative value of X to Y with a threshold value (ineffective groupthreshold value) comprising measuring the amount of CD80⁺ dendriticcells (X) and measuring the amount of a CD28⁺CD62L^(low)CD8⁺ T-cell (Y)as an indicator for predicting that the subject is not a part of anineffective group with respect to the cancer immunotherapy.

Since the inventors have discovered that multiple indicatorsindependently exhibit correlation with responsiveness, multipleindicators can be combined for use as an indicator for responsiveness.When two or more indicators are combined as an indicator forresponsiveness, an indicator represented by a formula using any numberof variables can be used. Examples of an indicator of responsivenessinclude, but are not limited to the following when multiple indicators(X₁, X₂, X₃, . . . X_(n)) are used:

F=a ₁ X ₁ ^(b1) +a ₂ X ₂ ^(b2) +a ₃ X ₃ ^(b3) . . . +a _(n) X _(n) ^(bn)

F=X ₁ ^(c1) *X ₂ ^(c2) *X ₃ ^(c3) . . . *X _(n) ^(cn)

wherein each of a, b, and c is any real number. Responsiveness can bepredicted from the difference derived from comparing a value calculatedby such a formula (indicator) with a threshold value. Multivariateanalysis (e.g., estimation by logistic regression) using discriminantanalysis on the novel indicator discovered by the inventors candetermine each coefficient for use as an indicator of responsiveness tocancer immunotherapy of a subject.

Typically, responsiveness can be predicted by formula F(X, Y) using twoindicators (X, Y) disclosed herein as variables. In a specificembodiment, the formula is a relative value of X to Y.

Any function (F(X, Y)) of X and Y can be used as the relative value of Xto Y. In particular, when X is considered to positively correlated withresponsiveness and Y is negatively correlated with responsiveness, anyfunction (F(X, Y)) of X and Y, which monotonically increases withrespect to X and monotonically decreases with respect to Y, can be used,but is not limited thereto. A formula indicating responsiveness with twoor more variables representing responsiveness can be found by regressionby calculating the contribution of each variable to responsiveness.

Examples of formula F(X, Y) indicating responsiveness include, but arenot limited to, the following:

F=aX ^(r) +bY ^(s)

F=X ^(r) *Y ^(s)

wherein a, b, r, and s are any real number.

For simplicity of the formula, an integer can be used as r and s. Insome embodiments, examples of relative values of X to Y include, but arenot limited to, X^(n)/Y^(m) (n and m are any real number such as anyinteger) such as X/Y and X²/Y. When factors X and Y each indicatesresponsiveness to therapy from different mechanisms, such a combinationof indicators can make prediction of responsiveness more accurate. Theinvestigation by the inventors demonstrated that responsiveness tocancer immunotherapy of a subject can be predicted more accurately usinga formula with r and s in the range of −5 to 5.

Another aspect of the present invention provides a method of furtherpredicting a subject who is a part of a response group (completeresponse (CR)+partial response (PR)) from among subjects who have beenshown to be not a part of an ineffective group by using the compositionof a CD4⁺ T-cells of the subjects as an indicator of responsiveness tocancer immunotherapy.

One embodiment of the present invention is a method of using a ratio ofa Foxp3⁺CD25⁺ T-cells in CD4⁺ T-cells, ICOS⁺CD62L^(low)CD4⁺ T cells inCD62L^(low)CD4⁺ T-cells, LAG-3⁺CD62L^(low)CD4⁺ T-cell subpopulation inCD62L^(low)CD4⁺ T-cells, or PD-1⁺CD62L^(low)CD4⁺ T-cell subpopulation inCD62L^(low)CD4⁺ T-cells in a subject, who is shown not to be a part ofan ineffective group, as an indicator of a response to cancerimmunotherapy of the subject. The ratio of Foxp3⁺CD25⁺ T-cells in CD4⁺T-cells, the ratio of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation inCD62L^(low)CD4⁺ T-cells, the ratio of LAG-3⁺CD62L^(low)CD4⁺ T-cellsubpopulation in CD62L^(low)CD4⁺ T-cells, or the ratio ofPD-1⁺CD62L^(low)CD4⁺ T-cell subpopulation in CD62L^(low)CD4⁺ T-cellshigher than an ineffective group threshold value can indicate that thesubject is not a part of a response group. To determine whether asubject is a part of a response group, it is necessary to determine thatthe subject is not a part of an ineffective group. Such a determinationof whether a subject is a part of an ineffective group can be achievedby a method disclosed herein.

Another embodiment of the present invention provides a method ofidentifying a response group (PR) and a stable group (SD) in apopulation of subjects determined not to be a part of an ineffectivegroup using the aforementioned (X, Y). In a method of identifying aresponse group (PR) and stable group (SD), variables (Z, W) can becalculated, with an amount of an ICOS⁺CD62L^(low)CD4⁺ T-cellsubpopulation as (Z) and a value selected from the group consisting of:

an amount of CD4⁺CD25⁺ T-cell subpopulation;an amount of CD4⁺Foxp3⁺ T-cell subpopulation;an amount of CD4⁺Foxp3⁺CD25⁺ T-cell subpopulation;an amount of CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation;an amount of CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation;an amount of CCR4⁺CD25⁺CD4⁺ T-cell subpopulation; andan amount of CD127⁺CD25⁺CD4⁺ T-cell subpopulation;as (W) to predict whether a subject is a part of the response group (PR)or stable group (SD). Typically, a method of identifying a responsegroup (PR) and a stable group (SD) can determine the response group (PR)and the stable group (SD) using the amount of an ICOS⁺CD62L^(low)CD4⁺T-cell subpopulation as (Z), the amount of a CD4⁺Foxp3⁺CD25⁺ T-cellsubpopulation as (W), and the value of W⁵*Z as an indicator.

A threshold value can be determined by considering sensitivity andspecificity. Sensitivity and specificity can be sensitivity andspecificity for the detection of an ineffective group, detection of aresponse group, or detection of a stable group. In one embodiment, athreshold value at which sensitivity and specificity are both 100% canbe set for the biomarker of the present invention. For this reason, anineffective case can be selected very accurately, so that this istechnically superior in a significant manner. When two or moreindicators disclosed as a biomarker of the present invention are used,threshold values can be determined for each indicator and used anddistinguished as a first threshold value, second threshold value, thirdthreshold value, and fourth threshold value as needed.

A threshold value can be determined so that sensitivity for thedetection of an ineffective group, detection of a response group, ordetection of a stable group exceeds about 90%. In another embodiment, athreshold value can be determined so that the sensitivity for thedetection of an ineffective group, detection of a response group, ordetection of a stable group is about 100%. In still another embodiment,a threshold value can be determined so that specificity for thedetection of an ineffective group, detection of a response group, ordetection of a stable group exceeds about 90%. In still anotherembodiment, a threshold value can be determined so that specificity forthe detection of an ineffective group, detection of a response group, ordetection of a stable group is about 100%.

In one embodiment, the composition of T-cells of a subject is acomposition of T-cells in a sample obtained from the subject.Preferably, a sample is a peripheral blood sample. The biomarkerprovided in the present invention can be measured using a peripheralblood sample, so that the biomarker has significant superiority inclinical applications, e.g., non-invasive, low-cost, and implementableover time.

In one embodiment, cancer immunotherapy comprises administration of animmune checkpoint inhibitor. In particular, the biomarker of the presentinvention can accurately predict a response to such cancer immunotherapyof a subject.

In one embodiment, an immune checkpoint inhibitor comprises a PD-1inhibitor or a PD-L1 inhibitor. Examples of PD-1 inhibitors include, butare not limited to, anti-PD-1 antibodies that inhibit an interactionbetween PD-1 and PD-L1 (e.g., binding) such as anti-PD-1 antibodiesnivolumab and pembrolizumab. Examples of PD-L1 inhibitors include, butare not limited to, anti-PD-L1 antibodies that inhibit an interactionbetween PD-1 and PD-L1 (e.g., binding) such as anti-PD-L1 antibodiesdurvalumab, atezolizumab, and avelumab.

Still another aspect of the present invention provides a method ofpredicting a response to cancer immunotherapy of a subject using thecomposition of T-cells of the subject to treat the subject with cancer.Alternatively, a method of treating cancer in a subject with a specificcomposition of T-cells or a composition therefor is provided. Cancerimmunotherapy, especially immune checkpoint inhibition therapy is knownto have a wide difference in responsiveness for each subject.Administration of cancer immunotherapy by selecting a subject using thebiomarker of the present invention can significantly increase theprobability of achieving a therapeutic effect such as tumor regression.

One embodiment of the present invention provides a method of treating asubject with cancer, comprising:

(1) determining a relative amount selected from the group consisting of:a relative amount of a CD4⁺ T-cell subpopulation correlated with adendritic cell stimulation in an anti-tumor immune response;a relative amount of a dendritic cell subpopulation correlated with adendritic cell stimulation by a CD4⁺ T-cell in an anti-tumor immuneresponse; anda relative amount of a CD8⁺ T-cell subpopulation correlated with adendritic cell stimulation in an anti-tumor immune response,in CD4⁺ T-cells in a sample derived from the subject; and(2) determining that the subject is not a part of an ineffective groupwith respect to a response to cancer immunotherapy if the relativeamount is higher than a threshold value (ineffective group thresholdvalue), and applying the cancer immunotherapy to the subject if thesubject is determined to be not a part of an ineffective group.

Another embodiment of the present invention provides a method oftreating a subject with cancer, comprising applying a cancerimmunotherapy to a subject if the subject is determined to be not a partof an ineffective group by determining a relative amount selected fromthe group consisting of:

a relative amount of a CD4⁺ T-cell subpopulation correlated with adendritic cell stimulation in an anti-tumor immune response;a relative amount of a dendritic cell subpopulation correlated with adendritic cell stimulation by a CD4⁺ T-cell in an anti-tumor immuneresponse; anda relative amount of a CD8⁺ T-cell subpopulation correlated with adendritic cell stimulation in an anti-tumor immune response,in CD4⁺ T-cells in a sample derived from the subject, and determiningthe relative amount to be higher than a threshold value (ineffectivegroup threshold value).

In this method, the relative amount is selected from the groupconsisting of:

a ratio of a CD62L^(low)CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CCR7⁻CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD45RA⁻CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD45RO⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of an LAG-3⁺CD62L^(low)CD4⁺ T-cell subpopulation inCD62L^(low)CD4⁺ T-cells;a ratio of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation inCD62L^(low)CD4⁺ T-cells;a ratio of a CCR4⁺CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD127⁺CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells,a ratio of a Foxp3⁺CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of an HLA-DR⁺ dendritic cell subpopulation in dendritic cells;a ratio of a CD80⁺ dendritic cell subpopulation in dendritic cells;a ratio of a CD86⁺ dendritic cell subpopulation in dendritic cells;a ratio of a PD-L1⁺ dendritic cell subpopulation in dendritic cells;a ratio of a CD62L^(low)CD8⁺ T-cell subpopulation in CD8⁺ T-cells;a ratio of a CD137⁺CD8⁺ T-cell subpopulation in CD8⁺ T-cells; anda ratio of a CD28⁺CD62L^(low)CD8⁺ T-cell subpopulation inCD62L^(low)CD8⁺ T-cells.

Preferably, the relative amount is selected from the group consistingof:

a ratio of a CD62L^(low)CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CCR7⁻CD4⁺ T-cell subpopulation in CD4⁺ T-cells,a ratio of a CD45RA⁻CD4⁺ T-cell subpopulation in CD4⁺ T-cells,a ratio of a CD45RO⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells,a ratio of an LAG-3⁺CD62L^(low)CD4⁺ T-cell subpopulation inCD62L^(low)CD4⁺ T-cells,a ratio of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation inCD62L^(low)CD4⁺ T-cells,a ratio of an HLA-DR⁺ dendritic cell subpopulation in dendritic cells,a ratio of a CD80⁺ dendritic cell subpopulation in dendritic cells,a ratio of a CD86⁺ dendritic cell subpopulation in dendritic cells,a ratio of a PD-L1⁺ dendritic cell subpopulation in dendritic cells,a ratio of a CD62L^(low)CD8⁺ T-cell subpopulation in CD8⁺ T-cells,a ratio of a CD137⁺CD8⁺ T-cell subpopulation in CD8⁺ T-cells, anda ratio of a CD28⁺CD62L^(low)CD8⁺ T-cell subpopulation inCD62L^(low)CD8⁺ T-cells.

Another embodiment of the present invention provides a method oftreating a subject with cancer, comprising: determining a ratio ofFoxp3⁺CD25⁺ T-cells in CD4⁺ T-cells in a sample derived from thesubject; determining that the subject is not a part of an ineffectivegroup with respect to a response to cancer immunotherapy if the ratio ofFoxp3⁺CD25⁺ T-cells in CD4⁺ T-cells is lower than a threshold value(ineffective group threshold value); and applying the cancerimmunotherapy to the subject if the subject is determined to be not apart of an ineffective group. Another embodiment of the presentinvention provides a method of treating a subject with cancer,comprising applying the cancer immunotherapy to the subject who isdetermined not a part of an ineffective group with respect to a responseto cancer immunotherapy by: determining a ratio of Foxp3⁺CD25⁺ T-cellsin CD4⁺ T-cells in a sample derived from the subject; and determiningthat the subject is not a part of an ineffective group with respect to aresponse to cancer immunotherapy if the ratio of Foxp3⁺CD25⁺ T-cells inCD4⁺ T-cells is lower than a threshold value (ineffective groupthreshold value).

Another embodiment of the present invention provides a method oftreating a subject with cancer, comprising:

(1) determining amounts (X, Y) selected from the group consisting of:a relative amount of a CD4⁺ T-cell subpopulation correlated with adendritic cell stimulation in an anti-tumor immune response;a relative of a dendritic cell subpopulation correlated with a dendriticcell stimulation by a CD4⁺ T-cell in an anti-tumor immune response;a relative of a CD8⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;an amount of regulatory T-cells or a CD4⁺ T-cell subpopulationcorrelated with regulatory T-cells; andan amount of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation;(2) using a comparison of a relative value of X to Y with an ineffectivegroup threshold value to determine whether the subject is a part of anineffective group with respect to a response to cancer immunotherapy;and(3) applying the cancer immunotherapy to the subject if the subject isdetermined to be not a part of an ineffective group.

Another embodiment of the present invention provides a method oftreating a subject with cancer, comprising applying the cancerimmunotherapy to the subject who is determined not a part of anineffective group with respect to a response to cancer immunotherapy by:

(1) determining amounts (X, Y) selected from the group consisting of:a relative amount of a CD4⁺ T-cell subpopulation correlated with adendritic cell stimulation in an anti-tumor immune response;a relative of a dendritic cell subpopulation correlated with a dendriticcell stimulation by a CD4⁺ T-cell in an anti-tumor immune response;a relative of a CD8⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;an amount of regulatory T-cells or a CD4⁺ T-cell subpopulationcorrelated with regulatory T-cells; andan amount of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation; and(2) using a comparison of a relative value of X to Y with an ineffectivegroup threshold value to determine whether the subject is a part of anineffective group with respect to a response to cancer immunotherapy.

For example, the aforementioned amounts (X) and (Y) are selected fromthe group consisting of:

an amount of a CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a CCR7⁻CD4⁺ T-cell subpopulation;an amount of an LAG-3⁺CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a CD45RA⁻CD4⁺ T-cell subpopulation;an amount of a CD45RO⁺CD4⁺ T-cell subpopulation;an amount of an HLA-DR⁺ dendritic cell subpopulation;an amount of a CD80⁺ dendritic cell subpopulation;an amount of a CD86⁺ dendritic cell subpopulation;an amount of a PD-L1⁺ dendritic cell subpopulation;an amount of a CD137⁺CD8⁺ T-cell subpopulation;an amount of a CD62L^(low)CD8⁺ T-cell subpopulation;an amount of a CD28⁺CD62L^(low)CD8⁺ T-cell subpopulation;an amount of a Foxp3⁺CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation;an amount of a CCR4⁺CD25⁺CD4⁺ T-cell subpopulation; andan amount of a CD127⁺CD25⁺CD4⁺ T-cell subpopulation.

For example, the method of the present invention can use a valueselected from the group consisting of:

an amount of a CD4⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;an amount of a dendritic cell subpopulation correlated with a dendriticcell stimulation by a CD4⁺ T-cell in an anti-tumor immune response; andan amount of a CD8⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;as (X). The method of the present invention can also calculate variables(X, Y), with a value selected from the group consisting of:an amount of a CD62L^(low) CD4⁺ T-cell subpopulation;an amount of a CCR7⁻CD4⁺ T-cell subpopulation;an amount of an LAG-3⁺CD62L^(low)CD4⁺ T-cell subpopulation;an amount of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a CD45RA⁻CD4⁺ T-cell subpopulation;an amount of a CD45RO⁺CD4⁺ T-cell subpopulation;an amount of an HLA-DR⁺ dendritic cell subpopulation;an amount of a CD80⁺ dendritic cell subpopulation;an amount of a CD86⁺ dendritic cell subpopulation;an amount of a PD-L1⁺ dendritic cell subpopulation;an amount of a CD62L^(low)CD8⁺ T-cell subpopulation;an amount of a CD137⁺CD8⁺ T-cell subpopulation; andan amount of a CD28⁺CD62L^(low)CD8⁺ T-cell subpopulation;

as (X).

For example, the method of the present invention can calculate variables(X, Y), with an amount of regulatory T-cells or a CD4⁺ T-cellsubpopulation correlated with regulatory T-cells as (Y). The method ofthe present invention can also calculate variables (X, Y), with a valueselected from the group consisting of:

an amount of a CCR4⁺CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD127⁺CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation; andan amount of a CD4⁺Foxp3⁺CD25⁺ T-cell subpopulation;

as (Y).

Another embodiment of the present invention provides a method oftreating a subject with cancer, comprising: determining amounts (X, Y)selected from the group consisting of:

an amount of a CD4⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;an amount of a dendritic cell subpopulation correlated with a dendriticcell stimulation by a CD4⁺ T-cell in an anti-tumor immune response;an amount of a CD8⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;an amount of regulatory T-cells or a CD4⁺ T-cell subpopulationcorrelated with regulatory T-cells; and an amount of anICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation; using a comparison of arelative value of X to Y with a threshold value (ineffective groupthreshold value) to determine whether the subject is a part of anineffective group with respect to a response to cancer immunotherapy;determining that the subject is a part of an effective group withrespect to a response to cancer immunotherapy if it is determined thatthe subject is not a part of an ineffective group, and a ratio ofFoxp3⁺CD25⁺ T-cells, a ratio of an ICOS⁺CD62L^(low)CD4⁺ T-cellsubpopulation, a ratio of LAG-3⁺CD62L^(low)CD4⁺ T-cell subpopulation, ora ratio of PD-1⁺CD62L^(low)CD4⁺ T-cell subpopulation is higher than athreshold value (effective group threshold value); and applying thecancer immunotherapy to the subject if the subject is determined to be apart of an effective group. Another embodiment of the present inventionprovides a method of treating a subject with cancer, comprising applyingthe cancer immunotherapy to the subject who is determined not a part ofan ineffective group with respect to a response to cancer immunotherapyby:determining amounts (X, Y) selected from the group consisting of:an amount of a CD4⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;an amount of a dendritic cell subpopulation correlated with a dendriticcell stimulation by a CD4⁺ T-cell in an anti-tumor immune response;an amount of a CD8⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;an amount of regulatory T-cells or a CD4⁺ T-cell subpopulationcorrelated with regulatory T-cells; and an amount of anICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation; using a comparison of arelative value of X to Y with a threshold value (ineffective groupthreshold value) to determine whether the subject is a part of anineffective group with respect to a response to cancer immunotherapy;determining that the subject is a part of an effective group withrespect to a response to cancer immunotherapy if it is determined thatthe subject is not a part of an ineffective group, and a ratio ofFoxp3⁺CD25⁺ T-cells, a ratio of an ICOS⁺CD62L^(low)CD4⁺ T-cellsubpopulation, a ratio of LAG-3⁺CD62L^(low)CD4⁺ T-cell subpopulation, ora ratio of PD-1⁺CD62L^(low)CD4⁺ T-cell subpopulation is higher than athreshold value (effective group threshold value).

For example, method of the present invention can use the value selectedfrom the group consisting of:

an amount of a CD4⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;an amount of a dendritic cell subpopulation correlated with a dendriticcell stimulation by a CD4⁺ T-cell in an anti-tumor immune response; andan amount of a CD8⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;as (X). The method of the present invention also can calculate variables(X, Y), with a value selected from the group consisting of:an amount of a CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a CCR7⁻CD4⁺ T-cell subpopulation;an amount of an LAG-3⁺CD62L^(low)CD4⁺ T-cell subpopulation;an amount of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a CD45RA⁻CD4⁺ T-cell subpopulation;an amount of a CD45RO⁺CD4⁺ T-cell subpopulation;an amount of an HLA-DR⁺ dendritic cell subpopulation;an amount of a CD80⁺ dendritic cell subpopulation;an amount of a CD86⁺ dendritic cell subpopulation;an amount of a PD-L1⁺ dendritic cell subpopulation;an amount of a CD62L^(low)CD8⁺ T-cell subpopulation; andan amount of a CD137⁺CD8⁺ T-cell subpopulation;an amount of a CD28⁺CD62L^(low)CD8⁺ T-cell subpopulation;

as (X).

For example, the method of the present invention can calculate variables(X, Y), with an amount of a regulatory T-cell subpopulation or a CD4⁺T-cell subpopulation correlated with regulatory T-cells as (Y). Themethod of the present invention can also calculate variables (X, Y),with a value selected from the group consisting of:

an amount of a CCR4⁺CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD127⁺CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation; andan amount of a CD4⁺Foxp3⁺CD25⁺ T-cell subpopulation;

as (Y).

Another aspect of the present invention provides a method of identifyinga response group (PR) and a stable group (SD) in a population ofsubjects determined to be not a part of an ineffective group using theaforementioned (X, Y). A method of identifying a response group (PR) anda stable group (SD) can calculate variables (Z, W), with an amount of anICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation as (Z) and a value selectedfrom the group consisting of:

an amount of a CD4⁺CD25⁺ T-cell subpopulation;an amount of a CD4⁺Foxp3⁺ T-cell subpopulation;an amount of a CD4⁺Foxp3⁺CD25⁺ T-cell subpopulation;an amount of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation;an amount of a CCR4⁺CD25⁺CD4⁺ T-cell subpopulation; andan amount of a CD127⁺CD25⁺CD4⁺ T-cell subpopulation;as (W) to predict whether a subject is a part of the response group (PR)or the stable group (SD).

Still another aspect of the present invention provides a kit forpredicting a response to cancer immunotherapy of a subject comprising anagent for detecting one or more cell surface markers selected from CD4,CD25, CD62L, Foxp3, and the like, such as a combination of markersselected from the group consisting of:

-   -   a combination of CD4 and CD62L;    -   a combination of CD4, CD45RA, and CCR7;    -   a combination of CD4, CD45RO, and CCR7;    -   a combination of CD4, CD62L, and LAG-3;    -   a combination of CD4, CD62L, and ICOS;    -   a combination of CD4, CD62L, and CD25;    -   a combination of CD4, CD127, and CD25;    -   a combination of CD4, CD45RA, and Foxp3;    -   a combination of CD4, CD45RO, and Foxp3;    -   a combination of CD4, CD25, and Foxp3;    -   a combination of CD11c, CD141, and HLA-DR;    -   a combination of CD11c, CD141, and CD80;    -   a combination of CD11c, CD123, and HLA-DR;    -   a combination of CD11c, CD123, and CD80;    -   a combination of CD8 and CD62L;    -   a combination of CD8 and CD137; and    -   a combination of CD28, CD62L and CD8. Preferably, the kit        comprises an agent for detecting each of CD4 and CD62L. A        combination of such detection agents can be used in determining        the composition of T-cells of a subject. Such a kit can be used        in measuring the ratio of a specific T-cell subpopulation as a        novel biomarker disclosed herein in a subject.

One embodiment of the present invention is a kit comprising an agent fordetecting a cell surface marker for predicting a response to cancerimmunotherapy of a subject. The inventors discovered that these cellsurface markers expressed by a T-cell of a subject are related toresponsiveness to cancer immunotherapy of a subject. It is understoodthat a kit comprising an agent for detecting such cell surface markersare useful in predicting responsiveness to cancer immunotherapy in viewof the above. A kit preferably comprises an agent for detecting CD4 andCD62L. A kit more preferably comprises an agent for detecting CD4, CD25,CD62L, and Foxp3. In one embodiment, a detection agent is an antibody.Preferably, an antibody facilitates the detection of a suitably labeledmarker.

Another aspect of the present invention is a composition for treatingcancer in a subject, comprising an immune checkpoint inhibitor.

One embodiment of the present invention is a composition for treatingcancer in a subject, comprising an immune checkpoint inhibitor,characterized in that the subject has a relative amount selected fromthe group consisting of:

a relative amount of a CD4⁺ T-cell subpopulation correlated with adendritic cell stimulation in an anti-tumor immune response;a relative amount of a dendritic cell subpopulation correlated with adendritic cell stimulation by a CD4⁺ T-cell in an anti-tumor immuneresponse; anda relative amount of a CD8⁺ T-cell subpopulation correlated with adendritic cell stimulation in an anti-tumor immune response;wherein the relative amount is equal to or greater than a thresholdvalue (ineffective group threshold value).

For example, the relative amount is typically selected from the groupconsisting of:

a ratio of a CD62L^(low)CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CCR7⁻CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a LAG-3⁺CD62L^(low)CD4⁺ T-cell subpopulation inCD62L^(low)CD4⁺ T-cells;a ratio of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation inCD62L^(low)CD4⁺ T-cells;a ratio of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD127⁺CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a Foxp3⁺CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of an HLA-DR⁺ dendritic cell subpopulation in dendritic cells;a ratio of a CD80⁺ dendritic cell subpopulation in dendritic cells;a ratio of a CD86⁺ dendritic cell subpopulation in dendritic cells;a ratio of a PD-L1⁺ dendritic cell subpopulation in dendritic cells;a ratio of a CD62L^(low)CD8⁺ T-cell subpopulation in CD8⁺ T-cells; anda ratio of a CD137⁺CD8⁺ T-cell subpopulation in CD8⁺ T-cells; anda ratio of a CD28⁺CD62L^(low)CD8⁺ T-cell subpopulation inCD62L^(low)CD8⁺ T-cells.

Still another embodiment of the present invention is a composition fortreating cancer in a subject, comprising an immune checkpoint inhibitor,characterized in that the subject is a subject selected by comparison ofa threshold value (ineffective group threshold value) and a relativevalue of X to Y with amounts (X, Y) selected from the group consistingof:

an amount of a CD4⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;an amount of a dendritic cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response; andan amount of a CD8⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;an amount of regulatory T-cells or a CD4⁺ T-cell subpopulationcorrelated with regulatory T-cells; and an ICOS⁺CD62L^(low)CD4⁺ T-cellsubpopulation;in a sample derived from the subject. The amounts (X, Y) are typicallyselected from the group consisting of:an amount of a CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a CCR7⁻CD4⁺ T-cell subpopulation;an amount of a LAG-3⁺CD62L^(low)CD4⁺ T-cell subpopulation;an amount of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a CCR4⁺CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD45RA⁻CD4⁺ T-cell subpopulation;an amount of a CD45RO⁺CD4⁺ T-cell subpopulation;an amount of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD127⁺CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation;an amount of a Foxp3⁺CD25⁺CD4⁺ T-cell subpopulation;an amount of an HLA-DR⁺ dendritic cell subpopulation;an amount of a CD80⁺ dendritic cell subpopulation;an amount of a CD86⁺ dendritic cell subpopulation;an amount of a PD-L1⁺ dendritic cell subpopulation;an amount of a CD62L^(low)CD8⁺ T-cell subpopulation;an amount of a CD137⁺CD8⁺ T-cell subpopulation; andan amount of a CD28⁺CD62L^(low)CD8⁺ T-cell subpopulation.

For example, the method of the present invention can use a valueselected from the group consisting of:

an amount of a CD4⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;an amount of a dendritic cell subpopulation correlated with a dendriticcell stimulation by a CD4⁺ T-cell in an anti-tumor immune response; andan amount of a CD8⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;as (X). The method of the present invention can also calculate variables(X, Y), with a value selected from the group consisting of:an amount of a CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a CCR7⁻CD4⁺ T-cell subpopulation;an amount of a LAG-3⁺CD62L^(low)CD4⁺ T-cell subpopulation;an amount of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a CD45RA⁻CD4⁺ T-cell subpopulation;an amount of a CD45RO⁺CD4⁺ T-cell subpopulation;an amount of an HLA-DR⁺ dendritic cell subpopulation;an amount of a CD80⁺ dendritic cell subpopulation;an amount of a CD86⁺ dendritic cell subpopulation;an amount of a PD-L1⁺ dendritic cell subpopulation;an amount of a CD62L^(low)CD8⁺ T-cell subpopulation;an amount of a CD137⁺CD8⁺ T-cell subpopulation; andan amount of a CD28⁺CD62L^(low)CD8⁺ T-cell subpopulation;

as (X).

For example, method of the present invention can calculate variables (X,Y), with the amount of regulatory T-cells or a CD4⁺ T-cell subpopulationcorrelated with regulatory T-cells as (Y). The method of the presentinvention can also calculate variables (X, Y), with a value selectedfrom the group consisting of:

an amount of a CCR4⁺CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD127⁺CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation; andan amount of a CD4⁺Foxp3⁺CD25⁺ T-cell subpopulation;

as (Y).

For example, the method of the present invention can use a comparison ofa relative value of X to Y with a threshold value (ineffective groupthreshold value), comprising measuring the amount of CD4⁺CD62L^(low)T-cells (X) and measuring the amount of CD4⁺Foxp3⁺CD25⁺ T-cells (Y) asan indicator for predicting that the subject is not a part of anineffective group with respect to the cancer immunotherapy. The amountor ratio of regulatory T-cells or a CD4⁺ T-cell subpopulation correlatedwith regulatory T-cells can be used as (Y).

Still another embodiment of the present invention is a composition fortreating cancer in a subject, comprising an immune checkpoint inhibitor,characterized in that the subject is a subject selected by comparison ofa threshold value (ineffective group threshold value) with a relativevalue of amounts (X, Y) selected from the group consisting of:

an amount of a CD4⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;an amount of a dendritic cell subpopulation correlated with a dendriticcell stimulation by a CD4⁺ T-cell in an anti-tumor immune response;an amount of a CD8⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;an amount of regulatory T-cells or a CD4⁺ T-cell subpopulationcorrelated with regulatory T-cells; and an amount of anICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation, in a sample derived from thesubject, and having a ratio of a Foxp3⁺CD25⁺ T-cell subpopulation inCD4⁺ T-cells or a ratio of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulationin CD62L^(low)CD4⁺ T-cells equal to or greater than a threshold value(effective group threshold value). The amounts (X) and (Y) are typicallyselected from the group consisting of:an amount of a CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a CCR7⁻CD4⁺ T-cell subpopulation;an amount of a LAG-3⁺CD62L^(low)CD4⁺ T-cell subpopulation;an amount of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a CCR4⁺CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD45RA⁻CD4⁺ T-cell subpopulation;an amount of a CD45RO⁺CD4⁺ T-cell subpopulation;an amount of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD127⁺CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation;an amount of a Foxp3⁺CD25⁺CD4⁺ T-cell subpopulation;an amount of an HLA-DR⁺ dendritic cell subpopulation;an amount of a CD80⁺ dendritic cell subpopulation;an amount of a CD86⁺ dendritic cell subpopulation;an amount of a PD-L1⁺ dendritic cell subpopulation;an amount of a CD62L^(low)CD8⁺ T-cell subpopulation;an amount of a CD137⁺CD8⁺ T-cell subpopulation; andan amount of a CD28⁺CD62L^(low)CD8⁺ T-cell subpopulation.

For example, the target of administration of the composition of thepresent invention can be a subject characterized by variables (X, Y),with a value selected from the group consisting of:

an amount of a CD4⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;an amount of a dendritic cell subpopulation correlated with a dendriticcell stimulation by a CD4⁺ T-cell in an anti-tumor immune response; andan amount of a CD8⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;as (X). The method of the present invention can also calculate variables(X, Y), with a value selected from the group consisting of:an amount of a CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a CCR7⁻ CD4⁺ T-cell subpopulation;an amount of a LAG-3⁺CD62L^(low)CD4⁺ T-cell subpopulation;an amount of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a CD45RA⁻CD4⁺ T-cell subpopulation;an amount of a CD45RO⁺CD4⁺ T-cell subpopulation;an amount of an HLA-DR⁺ dendritic cell subpopulation;an amount of a CD80⁺ dendritic cell subpopulation;an amount of a CD86⁺ dendritic cell subpopulation;an amount of a PD-L1⁺ dendritic cell subpopulation;an amount of a CD62L^(low)CD8⁺ T-cell subpopulation;an amount of a CD137⁺CD8⁺ T-cell subpopulation; andan amount of a CD28⁺CD62L^(low)CD8⁺ T-cell subpopulation;as (X) to target administration to a subject characterized by variables(X, Y).

For example, for the composition of the present invention, variables (X,Y) can be calculated, with an amount of regulatory T-cells or a CD4⁺T-cell subpopulation correlated with regulatory T-cells as (Y). Themethod of the present invention can also calculate variables (X, Y),with a value selected from the group consisting of:

an amount of a CCR4⁺CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD127⁺CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation; andan amount of a CD4⁺Foxp3⁺CD25⁺ T-cell subpopulation;as (Y) to target administration to a subject characterized by variables(X, Y).

For example, a subject predicted to be not a part of an ineffectivegroup with respect to cancer immunotherapy can be targeted as the targetof administrating the composition of the present invention by comparinga relative value of X to Y with a threshold value (ineffective groupthreshold value) from the amount of CD4⁺CD62L^(low) T-cells (X) and theamount of CD4⁺Foxp3⁺CD25⁺ T-cells (Y). The amount or ratio of regulatoryT-cells or a CD4⁺ T-cell subpopulation correlated with regulatoryT-cells can be used as (Y). The composition of the present invention canbe used in combination with any other agents.

In one embodiment, a composition comprises a PD-1 inhibitor. Examples ofPD-1 inhibitors include anti-PD-1 antibodies that inhibit a bindingbetween PD-L1 and PD-1, such as nivolumab or pembrolizumab. In anotherembodiment, the composition comprises a PD-L1 inhibitor. Examples ofPD-L1 inhibitors include anti-PD-L1 antibodies that inhibit a bindingbetween PD-L1 and PD-1, such as durvalumab, atezolizumab, and avelumab.Compositions comprising such immune checkpoint inhibitors are understoodas attaining a therapeutic effect at an especially high probability whenadministered to a subject selected using the biomarker of the presentinvention.

Another aspect of the present invention provides a method of improvingor maintaining responsiveness to cancer immunotherapy of a subject. Itwas discovered that cells selected from the group consisting of:

CD62L^(low)CD4⁺ T-cells;CCR7⁻CD4⁺ T-cells;LAG-3⁺CD62L^(low)CD4⁺ T-cells;ICOS⁺CD62L^(low)CD4⁺ T-cells;CCR4⁺CD25⁺CD4⁺ T-cells;CD45RA⁻CD4⁺ T-cells;CD45RO⁺CD4⁺ T-cells;CD62L^(high)CD25⁺CD4⁺ T-cells;CD127⁺CD25⁺CD4⁺ T-cells;CD45RA⁻Foxp3⁺CD4⁺ T-cells;Foxp3⁺CD25⁺CD4⁺ T-cells;HLA-DR⁺ dendritic cells;CD80⁺ dendritic cells;CD86⁺ dendritic cells;PD-L1⁺ dendritic cells;CD62L^(low)CD8⁺ T-cells;CD137⁺CD8⁺ T-cells; andCD28⁺CD62L^(low)CD8⁺ T-cells;are important to the response of a subject to cancer immunotherapy. Itis understood that use of such T-cells can improve or maintain theresponsiveness to cancer immunotherapy of a subject. One embodiment ofthe present invention is a composition comprising a CD62L^(low)CD4⁺T-cell. A CD62L^(low)CD4⁺ T-cell or a composition comprising the same isuseful for treating or preventing cancer and can be used in combinationwith cancer immunotherapy. In a still another embodiment, thecomposition may comprise a CD62L^(low)CD8⁺ T-cell in addition toCD62L^(low)CD4⁺ T-cell or the like.

One embodiment of the present invention provides a method of makingcancer immunotherapy effective in a subject for whom the cancerimmunotherapy is predicted to be ineffective by using a cell selectedfrom the group consisting of:

CD62L^(low)CD4⁺ T-cells;CCR7⁻CD4⁺ T-cells;LAG-3⁺CD62L^(low)CD4⁺ T-cells;ICOS⁺CD62L^(low)CD4⁺ T-cells;CCR4⁺CD25⁺CD4⁺ T-cells;CD45RA⁻CD4⁺ T-cells;CD45RO⁺CD4⁺ T-cells;CD62L^(high)CD25⁺CD4⁺ T-cells;CD127⁺CD25⁺CD4⁺ T-cells;CD45RA⁻Foxp3⁺CD4⁺ T-cells;Foxp3⁺CD25⁺CD4⁺ T-cells;HLA-DR⁺ dendritic cells;CD80⁺ dendritic cells;CD86⁺ dendritic cells;PD-L1⁺ dendritic cells;CD62L^(low)CD8⁺ T-cells;CD137⁺CD8⁺ T-cells; andCD28⁺CD62L^(low)CD8⁺ T-cells;or a composition therefor. Another embodiment of the present inventionprovides a method for sustaining an effect of cancer immunotherapy byusing, for example, CD62L^(low)CD4⁺ T-cells or a composition thereof.The results of the inventors' study, which elucidated thatCD62L^(low)CD4⁺ T-cells of peripheral blood play the role of anaccelerator for an anti-tumor immune response in cancer immunotherapy(especially anti-PD-1 antibody therapy and/or anti-PD-L1 antibodytherapy), is a novel finding, which provides a new and revolutionaryapproach to cancer therapy. The inventors have also discovered that acell selected from the group consisting of:CCR7⁻CD4⁺ T-cells;LAG-3⁺CD62L^(low)CD4⁺ T-cells;ICOS⁺CD62L^(low)CD4⁺ T-cells;CCR4⁺CD25⁺CD4⁺ T-cells;CD45RA⁻CD4⁺ T-cells;CD45RO⁺CD4⁺ T-cells;CD62L^(high)CD25⁺CD4⁺ T-cells;CD127⁺CD25⁺CD4⁺ T-cells;CD45RA⁻Foxp3⁺CD4⁺ T-cells;Foxp3⁺CD25⁺CD4⁺ T-cells;HLA-DR⁺ dendritic cells;CD80⁺ dendritic cells;CD86⁺ dendritic cells;PD-L1⁺ dendritic cells;CD62L^(low)CD8⁺ T-cells;CD137⁺CD8⁺ T-cells; andCD28⁺CD62L^(low)CD8⁺ T-cellsalso promote anti-tumor immune responses as in the CD62L^(low)CD4⁺T-cell disclosed above. In still another embodiment of the presentinvention, CD62L^(low)CD8⁺ T-cells may be used in addition toCD62L^(low)CD4⁺ T-cells.

Some embodiments of the present invention provide a method of refiningor purifying CD62L^(low)CD4⁺ T-cells or a method of manufacturing acomposition comprising CD62L^(low)CD4⁺ T-cells. In some embodiments,CD62L^(low)CD4⁺ T cells are isolated from a human derived sample. Someembodiments provide a method of preparing CD62L^(low)CD4⁺ T cells thathave been isolated from a subject for infusion into the subject and amethod of infusing such cells into the subject. One embodiment of thepresent invention is a composition comprising a CD62L^(low)CD4⁺ T-cell,which is from a subject to whom the composition is administered. A stillanother aspect of the present invention provides a method of refining orpurifying CD62L^(low)CD8⁺ T-cells or a method of manufacturing acomposition comprising CD62L^(low)CD8⁺ T-cells. Using the sameprinciple, the present invention similarly prepares a cell selected fromthe group consisting of:

CCR7⁻CD4⁺ T-cells;LAG-3⁺CD62L^(low)CD4⁺ T-cells;ICOS⁺CD62L^(low)CD4⁺ T-cells;CCR4⁺CD25⁺CD4⁺ T-cells;CD45RA⁻CD4⁺ T-cells;CD45RO⁺CD4⁺ T-cells;CD62L^(high)CD25⁺CD4⁺ T-cells;CD127⁺CD25⁺CD4⁺ T-cells;CD45RA⁻Foxp3⁺CD4⁺ T-cells;Foxp3⁺CD25⁺CD4⁺ T-cells;HLA-DR⁺ dendritic cells;CD80⁺ dendritic cells;CD86⁺ dendritic cells;PD-L1⁺ dendritic cells;CD62L^(low)CD8⁺ T-cells;CD137⁺CD8⁺ T-cells; andCD28⁺CD62L^(low)CD8⁺ T-cells.

A method of manufacturing a composition comprising CD62L^(low)CD4⁺T-cells can comprise purifying CD62L^(low)CD4⁺ T-cells from a T-cellpopulation derived from a human. The purifying may comprise removing aCD62L high expression cell from the T-cell population (negativeselection) Purification of CD62L^(low)CD4⁺ T-cells by negative selectionusing an antibody and/or magnetic beads and/or affinity column, or thelike is preferable because impurities such as an antibody or magneticbeads do not remain on a cell to be used. The present invention alsoprovides a method of manufacturing a composition comprisingCD62L^(low)CD8⁺ T-cells.

One embodiment of the present invention is a kit comprising a substance,which specifically binds to CD62L, for purifying CD62L^(low)CD4⁺T-cells. Examples of a substance which specifically binds to CD62Linclude, but are not limited to, antibodies that are specific to CD62L.

(Biomarkers of the Present Invention)

The biomarker of the present invention is understood to evaluate theoverall balance of anti-tumor immune responses, including CD4+ T-cells,dendritic cells, and/or CD8+ T-cells to evaluate the overall tumorimmunity itself. For this reason, the method of the present inventioncan be considered effective against a wide range of cancers and tumors.The present invention evaluates the overall anti-tumor immune responses,so that the present invention is also expected to be effective for notonly an immune checkpoint inhibitor against PD-1/PD-L1, but alsoanticancer therapy which acts on other immune checkpoints.

The present invention can also use a marker, which is indicative of aneffector T-cell, such as CCR7−, instead of or in addition toCD62L^(low). Alternatively, CD45RA− and/or CD45RO+ can be used. Forexample, the ratio of a CD45RA⁻CD4⁺ T-cell subpopulation in CD4⁺ T-cellsand/or the ratio of CD45RO⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells canalso be used. It was revealed that expression of LAG3 and ICOS can alsobe used (added or substituted) in a similar manner to CD62L^(low). Itwas similarly discovered that CCR4 expression can also be used (added orsubstituted) in a similar manner to CD62L^(low).

Instead of (or in addition to) using CD4⁺ T-cells (CD62L^(low)CD4⁺T-cells) which were used in the Examples, the number/ratio of cellsexpressing HLA-DR and/or CD80 and/or CD86 in a myeloid dendritic cell(mDC) and/or plasmacytoid dendritic cell (pDC) population can also beused as an indicator. PD-L1 on dendritic cells is also understood to beavailable as the marker of the present invention.

Further, instead (or in addition to) of using CD4⁺ T-cells (CD62L^(low)CD4⁺ T-cells) which were used in the Examples, the number/ratio of cellsexpressing 4-1BB in CD8+ T-cells can also be used as an indicator.

(Mechanism of the Present Invention)

Although not wishing to be bound by any theory, the anti-tumor immuneresponse phenomenon at a local tumor proposed by the inventors isschematically shown in FIG. 21. FIG. 21 shows cells that can be observedin peripheral blood, i.e., CD62L^(low)CD4⁺ T-cells, myeloid dendriticcells (mDC), plasmacytoid dendritic cells (pDC), and CD62L^(low)CD8⁺T-cells, and marker molecules expressed in these cells, i.e., LAG-3,ICOS, HLA-DR, CD80, and CD137. PD-L1 is expressed in dendritic cells,and PD-1 is expressed in CD62L^(low)CD4⁺ T-cells and CD62L^(low)CD8⁺T-cells.

The composition of T-cells is considered important in anti-tumor immuneresponses. For example, a stimulation of a dendritic cell by aCD62L^(low)CD4⁺ T-cell is important. If there are insufficientCD62L^(low)CD4⁺ T-cells (e.g., the balance of effector T-cells and naiveT-cells leans towards naive T-cells), dendritic cells cannot besufficiently stimulated even with administration of an immune checkpointinhibitor. As a result, an anti-tumor immune response cannot besufficient. For this reason, the ratio of CD62L^(low)CD4⁺ T-cells inCD4⁺ T-cells is an indicator for predicting an anti-tumor effect due toan immune checkpoint inhibitor. As with CD62L, the ratio of CD45RA⁻CCR7⁻ T-cells in CD4⁺ T-cells also indicates the balance of effectorT-cells and naive T-cells, so that such a ratio can be used as anindicator of the present invention.

Dendritic cells are stimulated by CD4⁺ T-cells via HLA-DR. Thus, with adecrease in the ratio of HLA-DR+ cells in dendritic cells, the dendriticcells cannot be sufficiently stimulated even with administration of animmune checkpoint inhibitor. As a result, anti-tumor immune responsescannot be sufficient. For this reason, the ratio of HLA-DR+ cells indendritic cells can also be an indicator for predicting an anti-tumoreffect due to an immune checkpoint inhibitor.

Dendritic cells which have been stimulated by CD4⁺ T-cells stimulateCD8⁺ T-cells, and stimulated CD8⁺ T-cells ultimately exert anti-tumoractivity. CD8⁺ T-cells are stimulated by dendritic cells via CD80/CD86expressed on dendritic cells and CD137 on CD8⁺ T-cells. Thus, both theratio of CD80+ cells in dendritic cells and the ratio of CD137+ cells inCD8⁺ T-cells can be an indicator for predicting an anti-tumor effect dueto an immune checkpoint inhibitor.

In addition to the biomarkers revealed from the mechanism disclosedabove, LAG-3, ICOS, and CCR4 in CD4⁺ T-cells were also found to be anindicator for predicting an anti-tumor effect due to an immunecheckpoint inhibitor as disclosed in the Examples.

For example, the present invention provides the following items.

(Item 1)

A method of using a relative amount of a CD4⁺ T-cell subpopulationcorrelated with a dendritic cell stimulation in an anti-tumor immuneresponse as an indicator for predicting a response to cancerimmunotherapy of a subject, comprising determining the relative amountof the CD4⁺ T-cell subpopulation in a sample derived from the subject,the relative amount higher than an ineffective group threshold valueindicating that the subject is not a part of an ineffective group to thecancer immunotherapy.

(Item 2)

The method of item 1, wherein the relative amount of the CD4⁺ T-cellsubpopulation is selected from the group consisting of:

a ratio of a CD62L^(low)CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CCR7⁻CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a LAG-3⁺CD62L^(low)CD4⁺ T-cell subpopulation inCD62L^(low)CD4⁺ T-cells;a ratio of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation inCD62L^(low)CD4⁺ T-cells;a ratio of a CCR4⁺CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD127⁺CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells; anda ratio of a Foxp3⁺CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells.

(Item 3)

A method of using a ratio of CD62L^(low) T-cells in CD4⁺ T-cells of asubject as an indicator for predicting a response to cancerimmunotherapy of the subject, comprising determining the ratio ofCD62L^(low) T-cells in the CD4⁺ T-cells in a sample derived from thesubject, the ratio higher than an ineffective group threshold valueindicating that the subject is not a part of an ineffective group to thecancer immunotherapy.

(Item 4)

A method of using a relative amount of a dendritic cell subpopulationcorrelated with a dendritic cell stimulation in an anti-tumor immuneresponse as an indicator for predicting a response to cancerimmunotherapy of a subject, comprising determining a ratio of thedendritic cell subpopulation in dendritic cells in a sample derived fromthe subject, the ratio higher than an ineffective group threshold valueindicating that the subject is not a part of an ineffective group to thecancer immunotherapy.

(Item 5)

The method of item 4, wherein the dendritic cell subpopulation isselected from the group consisting of: an HLA-DR⁺ dendritic cellsubpopulation; a CD80⁺ dendritic cell subpopulation; a CD86⁺ dendriticcell subpopulation; and PD-L1⁺ dendritic cell subpopulation.

(Item 6)

A method of using a relative amount of a CD8⁺ T-cell subpopulationcorrelated with a dendritic cell stimulation in an anti-tumor immuneresponse as an indicator for predicting a response to cancerimmunotherapy of a subject, comprising determining a ratio of the CD8⁺T-cell subpopulation in CD8⁺ T-cells in a sample derived from thesubject, the ratio higher than an ineffective group threshold valueindicating that the subject is not a part of an ineffective group to thecancer immunotherapy.

(Item 7)

The method of item 6, wherein the CD8⁺ T-cell subpopulation is aCD62L^(low)CD8⁺ T-cell subpopulation, a CD137⁺CD8⁺ T-cell subpopulation,or a CD28⁺CD62L^(low)CD8⁺ T-cell subpopulation.

(Item 8)

A method of using a relative value of amounts (X, Y) selected from thegroup consisting of:

an amount of a CD4⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;an amount of a dendritic cell subpopulation correlated with a dendriticcell stimulation by a CD4⁺ T-cell in an anti-tumor immune response;an amount of a CD8⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;an amount of regulatory T-cells or a CD4⁺ T-cell subpopulationcorrelated with regulatory T-cells; andan amount of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation;as an indicator for predicting a response to cancer immunotherapy of asubject, the method comprising:measuring the X; and measuring the Y;wherein a comparison of a relative value of X to Y with an ineffectivegroup threshold value is used as an indicator for predicting that thesubject is not a part of an ineffective group to the cancerimmunotherapy.

(Item 9)

The method of item 8, wherein the amounts (X) and (Y) are respectivelyselected from the group consisting of:

an amount of a CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a CCR7⁻CD4⁺ T-cell subpopulation;an amount of a LAG-3⁺CD62L^(low)CD4⁺ T-cell subpopulation;an amount of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a Foxp3⁺CD25⁺CD4⁺ T-cell subpopulation;an amount of an HLA-DR⁺ dendritic cell subpopulation;an amount of a CD80⁺ dendritic cell subpopulation;an amount of a CD86⁺ dendritic cell subpopulation;an amount of a PD-L1⁺ dendritic cell subpopulation;an amount of a CD62L^(low)CD8⁺ T-cell subpopulation;an amount of a CD137⁺CD8⁺ T-cell subpopulation; andan amount of a CD28⁺CD62L^(low)CD8⁺ T-cell subpopulation.

(Item 10)

The method of item 8 or 9, wherein the relative value is X/Y.

(Item 11)

The method of item 8 or 9, wherein the relative value is X²/Y.

(Item 12)

The method of any one of items 1 to 11, further using

a ratio of Foxp3⁺CD25⁺ T-cell subpopulation in CD4⁺ T-cells,a ratio of ICOS⁺CD62L^(low)CD4⁺ T cell subpopulation in CD62L^(low)CD4⁺T-cells,a ratio of LAG-3⁺CD62L^(low)CD4⁺ T cell subpopulation in CD62L^(low)CD4⁺T-cells, ora ratio of PD-1⁺CD62L^(low)CD4⁺ T cell subpopulation in CD62L^(low)CD4⁺T-cells, in a subject who is shown to be not a part of the ineffectivegroup as an indicator of a response to cancer immunotherapy of thesubject, whereinthe ratio of the Foxp3⁺CD25⁺ T-cell subpopulation in the CD4⁺ T-cells,the ratio of the ICOS⁺CD62L^(low)CD4⁺ T cell subpopulation in theCD62L^(low)CD4⁺ T-cellsthe ratio of LAG-3⁺CD62L^(low)CD4⁺ T cell subpopulation inCD62L^(low)CD4⁺ T-cells, orthe ratio of PD-1⁺CD62L^(low)CD4⁺ T cell subpopulation inCD62L^(low)CD4⁺ T-cells, higher than a response group threshold valueindicates that the subject is a part of a response group.

(Item 13)

The method of any one of items 1 to 12, wherein the ineffective groupthreshold value is determined by considering sensitivity and specificityfor the detection of an ineffective group.

(Item 14)

The method of any one of items 1 to 13, wherein the ineffective groupthreshold value is determined so that sensitivity for the detection ofan ineffective group exceeds about 90%.

(Item 15)

The method of any one of items 1 to 13, wherein the ineffective groupthreshold value is determined so that specificity for the detection ofan ineffective group exceeds about 90%.

(Item 16)

The method of any one of items 1 to 15, wherein the sample is aperipheral blood sample.

(Item 17)

The method of any one of items 1 to 16, wherein the cancer immunotherapycomprises administration of an immune checkpoint inhibitor.

(Item 18)

The method of item 17, wherein the immune checkpoint inhibitor isselected from the group consisting of a PD-1 inhibitor and a PD-L1inhibitor.

(Item 19)

The method of item 18, wherein the PD-1 inhibitor is an anti-PD-1antibody that inhibits an interaction between PD-1 and PD-L1.

(Item 20)

The method of item 18, wherein the PD-L1 inhibitor is an anti-PD-L1antibody that inhibits an interaction between PD-1 and PD-L1.

(Item 21)

The method of item 18, wherein the PD-1 inhibitor or PD-L1 inhibitorcomprises nivolumab, pembrolizumab, durvalumab, atezolizumab, oravelumab.

(Item 22)

A method of treating a subject with cancer, comprising:

(1) determining a relative amount selected from the group consisting of:a relative amount of a CD4⁺ T-cell subpopulation correlated with adendritic cell stimulation in an anti-tumor immune response;a relative amount of a dendritic cell subpopulation correlated with adendritic cell stimulation by a CD4⁺ T-cell in an anti-tumor immuneresponse; anda relative amount of a CD8⁺ T-cell subpopulation correlated with adendritic cell stimulation in an anti-tumor immune response;(2) determining that the subject is not a part of an ineffective groupwith respect to a response to cancer immunotherapy if the relativeamount is higher than an ineffective group threshold value; and(3) applying the cancer immunotherapy to the subject if the subject isdetermined to be not a part of an ineffective group.

(Item 23)

The method of item 22, wherein the relative amount is selected from thegroup consisting of:

a ratio of a CD62L^(low)CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CCR7⁻CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a LAG-3⁺CD62L^(low) CD4⁺ T-cell subpopulation inCD62L^(low)CD4⁺ T-cells;a ratio of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation inCD62L^(low)CD4⁺ T-cells;a ratio of a CCR4⁺CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD127⁺CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a Foxp3⁺CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of an HLA-DR⁺ dendritic cell subpopulation in dendritic cells;a ratio of a CD80⁺ dendritic cell subpopulation in dendritic cells;a ratio of a CD86⁺ dendritic cell subpopulation in dendritic cells;a ratio of a PD-L1⁺ dendritic cell subpopulation in dendritic cells;a ratio of a CD62L^(low)CD8⁺ T-cell subpopulation in CD8⁺ T-cells;a ratio of a CD137⁺CD8⁺ T-cell subpopulation in CD8⁺ T-cells; andan ratio of a CD28⁺CD62L^(low)CD8⁺ T-cell subpopulation inCD62L^(low)CD8⁺ T-cells.

(Item 24)

A method of treating a subject with cancer, comprising:

determining amounts (X, Y) selected from the group consisting of:an amount of a CD4⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;an amount of a dendritic cell subpopulation correlated with a dendriticcell stimulation by a CD4⁺ T-cell in an anti-tumor immune response;an amount of a CD8⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;an amount of regulatory T-cells or a CD4⁺ T-cell subpopulationcorrelated with regulatory T-cells; andan amount of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation;using a comparison of a relative value of X to Y with an ineffectivegroup threshold value to determine whether the subject is a part of anineffective group with respect to a response to cancer immunotherapy;andapplying the cancer immunotherapy to the subject if the subject isdetermined to be not a part of an ineffective group.

(Item 25)

The method of item 24, wherein the amounts (X) and (Y) are respectivelyselected from the group consisting of:

an amount of a CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a CCR7⁻CD4⁺ T-cell subpopulation;an amount of a LAG-3⁺CD62L^(low)CD4⁺ T-cell subpopulation;an amount of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a Foxp3⁺CD25⁺CD4⁺ T-cell subpopulation;an amount of an HLA-DR⁺ dendritic cell subpopulation;an amount of a CD80⁺ dendritic cell subpopulation;an amount of a CD86⁺ dendritic cell subpopulation;an amount of a PD-L1⁺ dendritic cell subpopulation;an amount of a CD62L^(low)CD8⁺ T-cell subpopulation; andan amount of a CD137⁺CD8⁺ T-cell subpopulation; andan amount of a CD28⁺CD62L^(low)CD8⁺ T-cell subpopulation.

(Item 26)

A method of treating a subject with cancer, comprising:

determining amounts (X, Y) selected from the group consisting of:an amount of a CD4⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;an amount of a dendritic cell subpopulation correlated with a dendriticcell stimulation by a CD4⁺ T-cell in an anti-tumor immune response;an amount of a CD8⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;an amount of regulatory T-cells or a CD4⁺ T-cell subpopulationcorrelated with regulatory T-cells; and an amount of anICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation; using a comparison of arelative value of X to Y with an ineffective group threshold value todetermine whether the subject is a part of an ineffective group withrespect to a response;determining that the subject is a part of a response group with respectto a response to cancer immunotherapy if it is determined that thesubject is not a part of an ineffective group, and a ratio of aFoxp3⁺CD25⁺ T-cell subpopulation, a ratio of an ICOS⁺CD62L^(low)CD4⁺T-cell subpopulation, a ratio of LAG-3⁺CD62L^(low)CD4⁺ T cellsubpopulation, or a ratio of PD-1⁺CD62L^(low)CD4⁺ T cell subpopulationis higher than a response group threshold value; andapplying the cancer immunotherapy to the subject if the subject isdetermined to be a part of a response group.

(Item 27)

A kit for predicting a response to cancer immunotherapy of a subject,comprising an agent for detecting a combination of markers selected fromthe group consisting of:

-   -   a combination of CD4 and CD62L;    -   a combination of CD4 and CCR7;    -   a combination of CD4, CD62L, and LAG-3;    -   a combination of CD4, CD62L, and ICOS;    -   a combination of CD4, CD62L, and CD25;    -   a combination of CD4, CD127, and CD25;    -   a combination of CD4, CD45RA, and Foxp3;    -   a combination of CD4, CD25, and Foxp3;    -   a combination of CD11c, CD141, and HLA-DR;    -   a combination of CD11c, CD141, and CD80;    -   a combination of CD11c, CD123, and HLA-DR;    -   a combination of CD11c, CD123, and CD80;    -   a combination of CD8 and CD62L;    -   a combination of CD8 and CD137; and    -   a combination of CD28, CD62L, and CD8.

(Item 28)

The kit of item 27, comprising an agent for detecting CD4 and CD62L.

(Item 29)

The kit of item 27, comprising an agent for detecting CD4, CD25, CD62L,and Foxp3.

(Item 30)

The kit of any one of items 27 to 29, wherein the detection agent is anantibody.

(Item 31)

A composition comprising an immune checkpoint inhibitor for treatingcancer in a subject, characterized in that the subject has a relativeamount selected from the group consisting of:

a relative amount of a CD4⁺ T-cell subpopulation correlated with adendritic cell stimulation in an anti-tumor immune response;a relative amount of a dendritic cell subpopulation correlated with adendritic cell stimulation by a CD4⁺ T-cell in an anti-tumor immuneresponse; anda relative amount of a CD8⁺ T-cell subpopulation correlated with adendritic cell stimulation in an anti-tumor immune response;wherein the relative amount is equal to or greater than an ineffectivegroup threshold value.

(Item 32)

The composition of item 31, wherein the relative amount is selected fromthe group consisting of:

a ratio of a CD62L^(low)CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CCR7⁻CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a LAG-3⁺CD62L^(low)CD4⁺ T-cell subpopulation inCD62L^(low)CD4⁺ T-cells;a ratio of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation inCD62L^(low)CD4⁺ T-cells;a ratio of a CCR4⁺CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD127⁺CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a Foxp3⁺CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of an HLA-DR⁺ dendritic cell subpopulation in dendritic cells;a ratio of a CD80⁺ dendritic cell subpopulation in dendritic cells;a ratio of a CD86⁺ dendritic cell subpopulation in dendritic cells;a ratio of a PD-L1⁺ dendritic cell subpopulation in dendritic cells;a ratio of a CD62L^(low)CD8⁺ T-cell subpopulation in CD8⁺ T-cells;a ratio of a CD137⁺CD8⁺ T-cell subpopulation in CD8⁺ T-cells; anda ratio of a CD28⁺CD62L^(low)CD8⁺ T-cell subpopulation inCD62L^(low)CD8⁺ T-cells.

(Item 33)

A composition comprising an immune checkpoint inhibitor for treatingcancer in a subject, characterized in that the subject is a subjectselected by comparison of an ineffective group threshold value with arelative value of amounts (X, Y) selected from the group consisting of:

an amount of a CD4⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;an amount of a dendritic cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response; andan amount of a CD8⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;an amount of regulatory T-cells or a CD4⁺ T-cell subpopulationcorrelated with regulatory T-cells; and an amount of anICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation.

(Item 34)

A composition comprising an immune checkpoint inhibitor for treatingcancer in a subject, characterized in that

the subject is a subject selected by comparison of an ineffective groupthreshold value with a relative value of amounts (X, Y) selected fromthe group consisting of:an amount of a CD4⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;an amount of a dendritic cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response; andan amount of a CD8⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;an amount of regulatory T-cells or a CD4⁺ T-cell subpopulationcorrelated with regulatory T-cells; and an amount of anICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation, andin a sample derived from the subject, havinga ratio of Foxp3⁺CD25⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of the ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation in theCD62L^(low) CD4⁺ T-cells;a ratio of LAG-3⁺CD62L^(low)CD4⁺ T cell subpopulation in CD62L^(low)CD4⁺T-cells; ora ratio of PD-1⁺CD62L^(low)CD4⁺ T cell subpopulation in CD62L^(low)CD4⁺T-cells being higher than a response group threshold value.

(Item 35)

The composition of any one of items 31 to 34, wherein the immunecheckpoint inhibitor is selected from the group consisting of a PD-1inhibitor and a PD-L1 inhibitor.

(Item 36)

The composition of item 35, wherein the PD-1 inhibitor is an anti-PD-L1antibody that inhibits an interaction between PD-L1 and PD-1.

(Item 37)

The composition of item 35, wherein the PD-L1 inhibitor is an anti-PD-L1antibody that inhibits an interaction between PD-1 and PD-L1.

(Item 38)

The composition of item 35, wherein the PD-1 inhibitor of PD-L1inhibitor is nivolumab, pembrolizumab, durvalumab, atezolizumab, oravelumab.

(Item 39)

A composition for treating or preventing cancer, comprising a cellselected from the group consisting of:

a CD62L^(low)CD4⁺ T-cell;a CCR7⁻CD4⁺ T-cell;a LAG-3⁺CD62L^(low)CD4⁺ T-cell;an ICOS⁺CD62L^(low)CD4⁺ T-cell;a CCR4⁺CD25⁺CD4⁺ T-cell;a CD62L^(high)CD25⁺CD4⁺ T-cell;a CD127⁺CD25⁺CD4⁺ T-cell;a CD45RA⁻Foxp3⁺CD4⁺ T-cell;a Foxp3⁺CD25⁺CD4⁺ T-cell;an HLA-DR⁺ dendritic cell;a CD80⁺ dendritic cell;a CD86⁺ dendritic cell;a PD-L1⁺ dendritic cell;a CD62L^(low)CD8⁺ T-cell;a CD137⁺CD8⁺ T-cell; anda CD28⁺CD62L^(low)CD8⁺ T-cell.

(Item 40)

The composition of item 39 for concomitant use with cancerimmunotherapy.

(Item 41)

The composition of item 39, characterized in that the composition isadministered in combination with an immune checkpoint inhibitor.

(Item 42)

The composition of item 41, wherein the immune checkpoint inhibitor isselected from the group consisting of a PD-1 inhibitor and a PD-L1inhibitor.

(Item 43)

The composition of item 42, wherein the PD-1 inhibitor is an anti-PD-1antibody that inhibits an interaction between PD-1 and PD-L1.

(Item 44)

The composition of item 42, wherein the PD-L1 inhibitor is an anti-PD-L1antibody that inhibits an interaction between PD-1 and PD-L1.

(Item 45)

The composition of item 42, wherein the PD-1 inhibitor or PD-L1inhibitor comprises nivolumab, pembrolizumab, durvalumab, atezolizumab,or avelumab.

(Item 46)

The composition of any one of items 39 to 45, further comprising aCD62L^(low)CD8⁺ T-cell.

(Item 47)

The composition of any one of items 39 to 46 for making cancerimmunotherapy effective in a subject to whom cancer immunotherapy ispredicted to be ineffective.

(Item 48)

The composition of any one of items 39 to 47 for sustaining an effect ofcancer immunotherapy.

(Item 49)

The composition of any one of items 39 to 48, wherein theCD62L^(low)CD4⁺ T-cell is from a subject to whom the composition isadministered.

(Item 50)

A method of manufacturing a composition for treating or preventingcancer comprising CD62L^(low)CD4⁺ T-cells, comprising purifyingCD62L^(low) CD4⁺ T-cells from a T-cell population derived from a human.

(Item 51)

The method of item 50, wherein the purifying comprises removing a CD62Lhigh expression cell from a T-cell population.

(Item 52)

A kit comprising a substance, which specifically binds to CD62L, forpurifying CD62L^(low) CD4⁺ T-cells.

(Item A1)

A method of using a ratio of CD62L^(low) T-cells in CD4⁺ T-cells of asubject as an indicator for predicting a response to cancerimmunotherapy of the subject, comprising determining the ratio ofCD62L^(low) T-cells in CD4⁺ T-cells in a sample derived from thesubject, the ratio higher than an ineffective group threshold valueindicating that the subject is not a part of an ineffective group to thecancer immunotherapy.

(Item A2)

A method of using a ratio of Foxp3⁺CD25⁺ T-cells in CD4⁺ T-cells of asubject as an indicator for predicting a response to cancerimmunotherapy of the subject, comprising determining the ratio ofFoxp3⁺CD25⁺ T-cells in CD4⁺ T-cells in a sample derived from thesubject, the ratio lower than an ineffective group threshold valueindicating that the subject is not a part of an ineffective group to thecancer immunotherapy.

(Item A3)

A method of using a relative value of an amount of CD4⁺CD62L^(low)T-cells of a subject (X) to an amount of CD4⁺Foxp3⁺CD25⁺ T-cells (Y) asan indicator for predicting a response to cancer immunotherapy of thesubject, the method comprising:

measuring the X; andmeasuring the Y;wherein a comparison of a relative value of X to Y with an ineffectivegroup threshold value is used as an indicator for predicting that thesubject is not a part of an ineffective group to the cancerimmunotherapy.

(Item A4)

The method of item A3, wherein the relative value is X/Y.

(Item A5)

The method of item A3, wherein the relative value is X²/Y.

(Item A6)

The method of any one of items A1 to A5, further using a ratio ofFoxp3⁺CD25⁺ T-cells in CD4⁺ T-cells in a subject who is shown not to bea part of the ineffective group as an indicator of a response to cancerimmunotherapy of the subject, wherein

the ratio of the Foxp3⁺CD25⁺CD4⁺ T-cells in the CD4⁺ T-cells higher thana response group threshold value indicates that the subject is a part ofa response group.

(Item A7)

The method of any one of items A1 to A6, wherein the ineffective groupthreshold value is determined by considering sensitivity and specificityfor the detection of an ineffective group.

(Item A8)

The method of any one of items A1 to A7, wherein the ineffective groupthreshold value is determined so that sensitivity for the detection ofan ineffective group exceeds about 90%.

(Item A9)

The method of any one of items A1 to A8, wherein the ineffective groupthreshold value is determined so that specificity for the detection ofan ineffective group exceeds about 90%.

(Item A10)

The method of any one of items A1 to A9, wherein the sample is aperipheral blood sample.

(Item A11)

The method of any one of items A1 to A10, wherein the cancerimmunotherapy comprises administration of an immune checkpointinhibitor.

(Item A12)

The method of item A11, wherein the immune checkpoint inhibitor isselected from the group consisting of a PD-1 inhibitor and a PD-L1inhibitor.

(Item A13)

The method of item A12, wherein the PD-1 inhibitor is an anti-PD-1antibody that inhibits an interaction between PD-1 and PD-L1.

(Item A14)

The method of item A12, wherein the PD-L1 inhibitor is an anti-PD-L1antibody that inhibits an interaction between PD-1 and PD-L1.

(Item A15)

The method of item A12, wherein the PD-1 inhibitor or PD-L1 inhibitorcomprises nivolumab, pembrolizumab, durvalumab, atezolizumab, oravelumab.

(Item A16)

A method of treating a subject with cancer, comprising:

determining a ratio of CD62L^(low) T-cells in CD4⁺ T-cells in a samplederived from the subject;determining that the subject is not a part of an ineffective group withrespect to a response to cancer immunotherapy if the ratio ofCD62L^(low) T-cells in CD4⁺ T-cells is higher than an ineffective groupthreshold value; andapplying the cancer immunotherapy to the subject if the subject isdetermined to be not a part of an ineffective group.

(Item A17)

A method of treating a subject with cancer, comprising:

determining a ratio of Foxp3⁺CD25⁺ T-cells in CD4⁺ T-cells in a samplederived from the subject;determining that the subject is not a part of an ineffective group withrespect to a response to cancer immunotherapy if the ratio ofFoxp3⁺CD25⁺ T-cells in CD4⁺ T-cells is lower than an ineffective groupthreshold value; andapplying the cancer immunotherapy to the subject if the subject isdetermined to be not a part of an ineffective group.

(Item A18)

A method of treating a subject with cancer, comprising:

determining an amount of CD4⁺CD62L^(low) T-cells (X);determining an amount of CD4⁺Foxp3⁺CD25⁺ T-cells (Y);using a comparison of a relative value of X to Y with an ineffectivegroup threshold value to determine whether the subject is a part of anineffective group with respect to a response to cancer immunotherapy;andapplying the cancer immunotherapy to the subject if the subject isdetermined to be not a part of an ineffective group.

(Item A19)

A method of treating a subject with cancer, comprising:

determining an amount of CD4+CD62L^(low) T-cells (X);determining an amount of CD4⁺Foxp3⁺CD25⁺ T-cells (Y);using a comparison of a relative value of X to Y with an ineffectivegroup threshold value to determine whether the subject is a part of anineffective group with respect to a response to cancer immunotherapy;determining that the subject is a part of a response group with respectto a response to cancer immunotherapy if the subject is determined to benot a part of an ineffective group and a ratio of Foxp3⁺CD25⁺ T-cells ishigher than a response group threshold value; andapplying the cancer immunotherapy to the subject if the subject isdetermined to be a part of a response group.

(Item A20)

A kit for predicting a response to cancer immunotherapy of a subject,comprising an agent for detecting one or more cell surface markersselected from CD4, CD25, CD62L, and Foxp3.

(Item A21)

The kit of item A20, comprising an agent for detecting CD4 and CD62L.

(Item A22)

The kit of item A20 or A21, comprising an agent for detecting CD4, CD25,CD62L, and Foxp3.

(Item A23)

The kit of any one of items A20 to 22, wherein the detection agent is anantibody.

(Item A24)

A composition comprising an immune checkpoint inhibitor for treatingcancer in a subject, characterized in that the subject has a ratio ofCD62L^(low)w T-cells in CD4+ cells in a sample derived from the subject,wherein the ratio is equal to or greater than an ineffective groupthreshold value.

(Item A25)

A composition comprising an immune checkpoint inhibitor for treatingcancer in a subject, characterized in that the subject has a ratio ofFoxp3⁺CD25⁺ T-cells in CD4+ cells in a sample derived from the subject,wherein the ratio is equal to or less than an ineffective groupthreshold value.

(Item A26)

A composition comprising an immune checkpoint inhibitor for treatingcancer in a subject, characterized in that the subject is a subjectselected by comparison of an ineffective group threshold value with arelative value of an amount of CD4⁺CD62L^(low) T-cells (X) in a samplederived from the subject to an amount of CD4⁺Foxp3⁺CD25⁺ T-cells (Y) ina sample derived from the subject.

(Item A27)

A composition comprising an immune checkpoint inhibitor for treatingcancer in a subject, characterized in that the subject is a subjectselected by comparison of an ineffective group threshold value with arelative value of an amount of CD4⁺CD62L^(low) T-cells (X) in a samplederived from the subject to an amount of CD4⁺Foxp3⁺CD25⁺ T-cells (Y),and in a sample derived from the subject, having a ratio of Foxp3⁺CD25⁺T-cell subpopulation in CD4⁺ T-cells being equal to or higher than aresponse group threshold value.

(Item A28)

The composition of any one of items A24 to A27, wherein the immunecheckpoint inhibitor is selected from the group consisting of a PD-1inhibitor and a PD-L1 inhibitor.

(Item A29)

The composition of item A28, wherein the PD-1 inhibitor is an anti-PD-1antibody that inhibits an interaction between PD-1 and PD-L1.

(Item A30)

The composition of item A28, wherein the PD-L1 inhibitor is ananti-PD-L1 antibody that inhibits an interaction between PD-1 and PD-L1.

(Item A31)

The composition of item A28, wherein the PD-1 inhibitor or PD-L1inhibitor comprises nivolumab, pembrolizumab, durvalumab, atezolizumab,or avelumab.

(Item A32)

A composition for treating or preventing cancer, comprising aCD62L^(low)CD4⁺ T-cell.

(Item A33)

The composition of item A32 for concomitant use with cancerimmunotherapy.

(Item A34)

The composition of item A32, characterized in that the composition isadministered in combination with an immune checkpoint inhibitor.

(Item A35)

The composition of items A34, wherein the immune checkpoint inhibitor isselected from the group consisting of a PD-1 inhibitor and a PD-L1inhibitor.

(Item A36)

The composition of item A35, wherein the PD-1 inhibitor is an anti-PD-1antibody that inhibits an interaction between PD-1 and PD-L1.

(Item A37)

The composition of item A35, wherein the PD-L1 inhibitor is ananti-PD-L1 antibody that inhibits an interaction between PD-1 and PD-L1.

(Item A38)

The composition of item A35, wherein the PD-1 inhibitor or PD-L1inhibitor comprises nivolumab, pembrolizumab, durvalumab, atezolizumab,or avelumab.

(Item A39)

The composition of any one of items A32 to A38, further comprising aCD62L^(low)CD8⁺ T-cell.

(Item A40)

The composition of any one of items A32 to A39 for making cancerimmunotherapy effective in a subject for whom the cancer immunotherapyis predicted to be ineffective.

(Item A41)

The composition of any one of items A32 to A40 for sustaining an effectof cancer immunotherapy.

(Item A42)

The composition of any one of items A32 to A41, wherein theCD62L^(low)CD4⁺ T-cell is from a subject to whom the composition isadministered.

(Item A43)

A method of manufacturing a composition for treating or preventingcancer comprising CD62L^(low)CD4⁺ T-cells, comprising purifyingCD62L^(low) CD4⁺ T-cells from a T-cell population derived from a human.

(Item A44)

The method of item A43, wherein the purifying comprises removing a CD62Lhigh expression cell from a T-cell population.

(Item A45)

A kit comprising a substance, which specifically binds to CD62L, forpurifying CD62L^(low)CD4⁺ T-cells.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram showing results of fractionation by flow cytometryof T-cells in a peripheral blood sample obtained from a subject. The topleft diagram identifies a lymphocyte region by two-dimensional analysisusing FSC and SSC. The top right diagram is a fraction with respect toCD8 and CD4 expression. The bottom left diagram is a fraction ofCD25⁺FoxP3⁺. The bottom right diagram is a histogram with respect toCD62L expression levels. CD62L low expression (CD62L^(low)) cells arefractionated in a double peak distribution.

FIG. 2 is a schematic diagram showing the procedure of measuring atherapeutic effect in Example 1.

FIGS. 3A-3D are diagrams comparing the cell count and T-cell compositionbetween an ineffective group and other groups. Panel A (WBC) comparesthe peripheral blood white blood cell (White Blood Cell) count. Panel B(Lym) compares the lymphocyte count. Panel C compares the percentage ofCD4+ cells. Panel D compares the percentage of CD8+ cells. A significantdifference was not found between the ineffective group and other groupswith respect to these parameters.

FIGS. 4A-4E are diagrams comparing the T-cell composition between anineffective group and other group. Panel A compares the percentage ofCD62L^(low) cells in CD8⁺ T-cells. This is significantly lower for a PDgroup. P=0.0138. Panel B compares the percentage of CD62L^(low) cells inCD4⁺ T-cells. A more significant decrease was observed in the PD groupthan the percentage of CD62L^(low) cells in CD8⁺ T-cells. P=5.32×10⁻⁷.Panel C compares the percentage of CD25⁺FoxP3⁺cells in CD4+ T-cells.This was significantly higher in the PD group. P=0.0132. Panel D is ascatter diagram for the percentage of CD62L^(low) cells in CD8⁺ T-cellsand the percentage of CD62L^(low) cells in CD4⁺ T-cells. A weekcorrelation was found between these values. Panel E is a scatter diagramof the percentage of CD62L^(low) cells in CD4⁺ T-cells and thepercentage of CD25⁺FoxP3⁺cells in CD4⁺ T-cells. A correlation was notfound between these values. It is understood that they eachindependently contribute to responsiveness to cancer immunotherapy.

FIG. 5 shows the performance of the percentage of CD62L^(low) cells inCD4⁺ T-cells as an indicator for distinguishing a PR+SD group from a PDgroup. The right panel plots the sensitivity and specificity uponchanges in a threshold value. The area of the region under the plottedpoints is 0.974. Thus, this is understood as a very good marker.

FIG. 6 shows the sensitivity and specificity of the percentage ofCD62L^(low) cells in CD4⁺ T-cells upon changing a threshold value fordistinguishing a PR+SD group from PD group.

FIG. 7 shows the performance of the relative value (X/Y) of thepercentage of CD62L^(low) cells in CD4⁺ T-cells (X) to the percentage ofCD25⁺FoxP3⁺cells in CD4⁺ T-cells (Y) as an indicator for distinguishinga PR+SD group from a PD group. The right panel plots the sensitivity andspecificity upon changing a threshold value. The area of the regionunder the plotted points is 0.961. Thus, this is understood as a verygood marker.

FIG. 8 shows the sensitivity and specificity when using X/Y as arelative value of the percentage of CD62L^(low) cells in CD4⁺ T-cells(X) to the percentage of CD25⁺FoxP3⁺cells in CD4⁺ T-cells upon changinga threshold value for distinguishing a PR+SD group from a PD group.

FIG. 9 shows the performance of a relative value (X²/Y) of thepercentage of CD62L^(low) cells in CD4⁺ T-cells (X) to the percentage ofCD25⁺FoxP3⁺cells in CD4⁺ T-cells as an indicator for distinguishing aPR+SD group from a PD group. The right panel is a plot of sensitivityand specificity upon changing a threshold value. The area of the regionunder the plotted points is 1.0, which shows that the indicator is avery advantageous marker enabling the determination at sensitivity andspecificity of 100%.

FIG. 10 shows the sensitivity and specificity when using X²/Y as arelative value of the percentage of CD62L^(low)w cells in CD4⁺ T-cells(X) to the percentage of CD25⁺FoxP3⁺cells in CD4⁺ T-cells upon changinga threshold value for distinguishing a PR+SD group from a PD group.

FIGS. 11A-11B show the performance of the percentage of CD25⁺FoxP3⁺cellsin CD4⁺ T-cells as an indicator for distinguishing a PR group and an SDgroup. The right panel is a plot of sensitivity and specificity uponchanging a threshold value. The area of the region under the plottedpoints is 0.773.

FIG. 12 shows the sensitivity and specificity of the percentage ofCD25⁺FoxP3⁺cells in CD4⁺ T-cells upon changing a threshold value fordistinguishing a PR group and an SD group.

FIG. 13 is a schematic diagram showing an example of an embodiment of amethod for improving or maintaining responsiveness to cancerimmunotherapy of a subject.

FIGS. 14A-14B are diagrams showing the relationship between thetherapeutic effect in mice subjected to T-cell infusion and the ratio of(CD62L^(low) cells in CD4⁺ T-cells)/(CD62L^(high)CD25⁺cells in CD4⁺T-cells). The horizontal axis indicates the number of days from tumorinoculation in mice. The composition of cells indicated by the label wasinfused. The vertical axis is the tumor size (mm). In the left panel,the ratio of (CD62L^(low) cells in CD4⁺ T-cells)/(CD62L^(high)CD25⁺cellsin CD4⁺ T-cells) in the spleen of mice subjected to cell infusion wasmeasured as of the time indicated by an arrow. The ratios had each ofthe values indicated in the bottom row. In the right panel, the ratio of(CD62L^(low) cells in CD4⁺ T-cells)/(CD62L^(high)CD25⁺cells in CD4⁺T-cells) in the spleen of mice was measured over time. The valueschanged as indicated in the bottom row.

FIGS. 15A-15D are diagrams showing CD62L staining patterns of differenthuman races and mice. Panel A shows FACS using lymph nodes drainingCaucasian tumor vaccine. The lymphocytes region was gated, and CD62L wasobserved. C is a similar observation of CD62L from peripheral bloodderived mononuclear cells of Japanese subjects. Panel D shows CD62Lstaining patterns in lymphocytes of mice. It is understood that similarstaining patterns are exhibited across human races/organism species.This has a double peak distribution, with fluorescence intensity of 10²as the boundary. Panel B is FACS showing the purity after separatingonly CD62L^(low) cells from the group of cells of a subject in panel Awith magnetic beads.

FIG. 16 is a graph showing the relationship between the ratio of CD80cells (top right) and the ratio of HLA-DR⁺cells (top left) in myeloiddendritic cells (mDC) and PD, SD, and PR, and a graph showing therelationship between the ratio of CD80 cells (bottom right) and theratio of HLA-DR⁺cells (bottom left) in plasmacytoid dendritic cells(pDC) and PD, SD, and PR.

FIG. 17 is a graph showing the correlation between the ratio of CD80cells (top right) and the ratio of HLA-DR⁺cells (top left) in myeloiddendritic cells (mDC) and CD62L^(low)CD4⁺ T-cells, and a graph showingthe correlation between the ratio of CD80 cells (bottom right) and theratio of HLA-DR⁺cells (bottom left) in plasmacytoid dendritic cells(pDC) and X²/Y (i.e., (amount of CD62L^(low)CD4⁺T-cells)²/(CD4⁺Foxp3⁺CD25⁺ T-cell)).

FIG. 18 is a result showing the ratio of CD80 cells (top right) and theratio of HLA-DR⁺cells (top left) in myeloid dendritic cells (mDC) andthe ratio of CD137⁺CD62L^(low) CD8⁺ T-cells to CD62L^(low)CD8CD62L^(low)CD8⁺ T-cells.

FIGS. 19A-19B are graphs showing the relationship between the ratio ofICOS⁺cells (FIG. 19B, right) and the ratio of LAG3⁺cells (FIG. 19A,left) in CD62L^(low)CD4⁺ T-cells and PD and PR+SD.

FIGS. 20A-20D are graphs showing the relationship between the ratio ofCXCR3⁺cells (FIG. 20A, top left), the ratio of CCR4⁺cells (FIG. 20B, topright), the ratio of CCR6⁺cells (FIG. 20C, bottom left), and the ratioof CXCR5⁺cells (FIG. 20D, bottom right) in CD62L^(low)CD4⁺ T-cells andPD and PR+SD. Only CCR4 exhibited a correlation that is sufficient as amarker (p=0.0250).

FIG. 21 is a graph showing the relationship between CD25⁺Foxp3⁺CD4⁺T-cells in CD4⁺ T-cells (top left) or ICOS⁺CD62L^(low)CD4⁺ T-cells inCD62L^(low)CD4⁺ T-cells (top right) and PR and SD. The bottom panelshows the sensitivity and specificity upon changing a threshold value ofW*Z for distinguishing a PR group and an SD group using the product W*Zof the ratio of CD25⁺Foxp3⁺CD4⁺ T-cells in CD4⁺ T-cells (W) and theratio of ICOS⁺CD62L^(low)CD4⁺ T-cells in CD62L^(low)CD4⁺ T-cells (Z).

FIG. 22 is a schematic diagram disclosing the mechanism associated withthe present invention.

FIG. 23 is a table showing antibodies used in the Examples.

FIG. 24 is a diagram showing logistic regression when using the shownbiomarkers alone for determination of a response group.

FIG. 25 is a diagram showing logistic regression for deriving a suitableformula in a combination of the ratio of CD25⁺Foxp3⁺CD4⁺ T-cells in CD4⁺T-cells (W) and the ratio of ICOS⁺CD62L^(low)CD4⁺ T-cells inCD62L^(low)CD4⁺ T-cells (Z) for determination of a response group.

FIG. 26 is a diagram showing results of ROC analysis when using aformula of a combination of the ratio of CD25⁺Foxp3⁺CD4⁺ T-cells in CD4⁺T-cells (W) and the ratio of ICOS⁺CD62L^(low)CD4⁺ T-cells inCD62L^(low)CD4⁺ T-cells (Z), which was found by logistic regression. Itis demonstrated that a response group can be determined with higherprecision compared to individual biomarkers by using said formula.

FIG. 27 is a diagram showing logistic regression for deriving a suitableformula in a combination of the percentage of CD62L^(low) cells in CD4⁺T-cells (X) and the percentage of CD25⁺FoxP3⁺in CD4⁺ T-cells (Y) fordetermination of an ineffective group.

FIG. 28 is a diagram showing one example of a method of setting athreshold value of CD62L^(low) and CD62L^(high).

FIGS. 29A-29B are histograms with respect to CD62L expression levels,which shows that CD62L low expression (CD62L^(low)) cells are clearlyseparated.

FIGS. 30a-30g are diagrams showing the prediction of treatment outcomein the discovery and validation cohorts. (a) Prediction formula valuesin the patient discovery cohort patients. Prediction formula, X²/Y, wasbased on the percentages of CD62L^(low) cells (X) and CD25⁺FOXP3⁺cells(Y) in the total population of CD4⁺cells. (b) The receiver operatingcharacteristic curve of the prediction formula that predictedNon-responders in the discovery cohort (n=40). The sensitivity andspecificity parameters at the threshold value of Prediction formula(192) were 85.7% and 100% (P<0.0001). (c) The progression-free survival(PFS) curves of the discovery cohort patients who were diagnosed asNon-responders or responders on the basis of the threshold value ofPrediction formula (192). (d) Overall survival (OS) curves of thediscovery cohort. (e) The values of the prediction formula in thevalidation cohort of patients. In these patients, peripheral bloodmononuclear cells were examined before CT evaluation. (f) PFS curves ofvalidation cohort patients. (g) OS curves of validation cohort patients.In panels a and e, data are presented as the mean±standard error of themean and symbols indicate values from individual patients. Statisticalsignificance of differences was assessed by the Student's two-tailedt-test (a,e) or log-rank test (b-d,f,g).

FIG. 31 is a diagram showing correlation between the percentage ofCD28⁺cells in the total population of CD62L^(low) CD8⁺ T-cells andprediction formula (X²/Y, wherein X=the ratio of CD62L^(low) T-cells inthe CD4⁺ T-cell population (%) and Y=the ratio of CD25⁺FOXP3⁺ T-cells inthe CD4⁺ T-cell population (%)) values (N=12).

FIGS. 32a-32f show differences in the percentages of T cellsub-populations and prediction formula values in patients with non-smallcell lung cancer with different treatment outcomes. FACS(Fluorescence-activated cell sorting) results from peripheral bloodsamples of three subgroups of patients (N=81 in total) who were goodresponders (GR), intermediate responders (IR), and non-responders (NR)at 8 weeks during the first tumor response evaluation after Nivolumabtreatment. The percentages of PD-1⁺, LAG-3⁺and ICOS⁺cells in the totalpopulation of CD62L^(low) CD4⁺cells and CD62L^(high)CD4⁺cells areindicated in d-f, respectively. Data are presented as the means±standarderror of the mean. Symbols indicate values from individual patients.Statistical significance of differences was assessed by one-way analysisof variance (ANOVA) and subsequent post hoc analysis (Two-stage step-upmethod of Benjamin, Krieger, and Yekutieli).

FIGS. 33a-33b are diagrams showing gene expression responsible for goodresponse to Nivolumab treatment. FIG. 33a is signatures obtained bycomparing gene expression data between CD62L^(high)CD4⁺and CD62L^(low)CD4⁺ T cells from good responders (GR), intermediate responders (IR),and non-responders (NR). In FIG. 33b , among 39 genes well-known to berelated to anti-tumor immunity in the above signatures, the geneexpression of 29 is shown in terms of Nivolumab-treatment response. Thedegree of expression of the genes in CD62L^(low) CD4⁺ T cells is shown,which indicates relatively higher gene expression in GR compared to IRand NR, and in GR and IR compared to NR, is depicted.

FIG. 34a shows immunity-related genes that showed differentialexpression between CD62L^(low) CD4+ T cells and CD62L^(high)CD4+ Tcells, commonly in good, intermediate, and non-responder patients. It isconsidered that the recited genes can be used for distinction of cellsubpopulations. FIG. 34b shows 53 genes that showed differentialexpressions related to the response to Nivolumab in CD62L^(low)CD4⁺ Tcells. It is understood that the recited genes can be used as markersfor distinction of patient groups by examining their expression onCD62L^(low)CD4⁺ T cells. Good responders: GR, intermediate responders:IR, and non-responders: NR.

FIG. 35 is a diagram showing the change of survival ratio in (1) Controlgroup, (2) Antibody group, and (3) Antibody+Cell group.

DESCRIPTION OF EMBODIMENTS

The present invention is disclosed hereinafter using exemplary Exampleswhile referring to the appended drawings as needed. Throughout theentire specification, a singular expression should be understood asencompassing the concept thereof in the plural form, unless specificallynoted otherwise. Further, the terms used herein should be understood tobe used in the meaning that is commonly used in the art, unlessspecifically noted otherwise. Thus, unless defined otherwise, allterminologies and scientific technical terms that are used herein havethe same meaning as the general understanding of those skilled in theart to which the present invention pertains. In case of a contradiction,the present specification (including the definitions) takes precedence.

Definitions

As used herein, “biomarker” refers to characteristics that can beobjectively measured and evaluated as an indicator of a normalbiological process, pathological process, or a pharmacological responseto therapeutic intervention.

As used herein, “cancer” refers to malignant tumor, which is highlyatypical, expands faster than normal cells, and can destructivelyinfiltrate or metastasize surrounding tissue, or the presence thereof.In the present invention, cancer includes, but is not limited to, solidcancer and hematopoietic tumor.

As used herein, “cancer immunotherapy” refers to a method of treatingcancer using a biological defense mechanism such as the immune mechanismof organisms.

As used herein, “anti-tumor immune response” refers to any immuneresponse against tumor in a live organism.

As used herein, “dendritic cell stimulation in an anti-tumor immuneresponse” refers to any phenomenon applying a stimulation to dendriticcells, which occurs in the process of an immune response against tumorin a live organism. Such stimulation can be a direct or indirect factorfor inducing an anti-tumor immune response. Although not limited to thefollowing, typically, a dendritic cell stimulation in an anti-tumorimmune response is applied by CD4⁺ T-cells (e.g., effector T-cells),resulting in dendritic cells stimulating CD8⁺ T-cells, and thestimulated CD8⁺ T-cells exerting an anti-tumor effect.

As used herein, “correlation” refers to two matters having astatistically significant correlated relationship For example, “relativeamount of B correlated with A” refers to the relative amount of B beingstatistically significantly affected (e.g., increase or decrease) when Aoccurs.

As used herein, “cell subpopulation” refers to a cell populationconstituting a part of an entire cell population.

As used herein, the term “relative amount” with regard to cells can beinterchangeably used with “ratio”. Typically, the terms “relativeamount” and “ratio” refer to the number of cells constituting a givencell subpopulation (e.g., CD62L^(low)CD4⁺ T-cells) with respect to thenumber of cells constituting a specific cell population (e.g., CD4⁺T-cell population).

As used herein, “sensitivity” refers to the ratio of number of subjectshaving a given feature in selected targets to the total number ofsubjects with the given feature in a subject population when selecting asubject with the given feature from among a population of subjects,i.e., (number of subjects having a given feature in a selectedtarget)/(total number of subjects with the given feature in a subjectpopulation).

As used herein, “specificity” refers to the ratio of the number ofsubjects with a given feature in selected targets to the total number ofselected targets when selecting a subject with the given feature fromamong a population of subjects, i.e., (number of subjects with a givenfeature among selected targets)/(total number of selected targets).

As used herein, “ineffective group” refers to a group of subjectsdetermined to be progressive (PD, Progressive disease) when atherapeutic effect upon undergoing cancer therapy is determinedaccording to RECIST ver 1.1 at the early stage of up to about 9 weeksafter start of the treatment. An ineffective group is also called a PDgroup, progressive group, or NR (Non-responder) which areinterchangeably used herein.

As used herein, “partial response group” refers to a group of subjectsdetermined to be partial response (PR, Partial response) when atherapeutic effect upon undergoing cancer therapy is determinedaccording to RECIST ver 1.1. A partial response group is also called aPR group, which is interchangeably used herein.

As used herein, “stable group” refers to a group of subjects determinedto be stable (SD, Stable disease) when a therapeutic effect uponundergoing cancer therapy is determined according to RECIST ver 1.1 atthe early stage of up to about 9 weeks after start of the treatment. A“stable group” is also called an SD group or intermediate group, whichare interchangeably used herein. Further, once the group turns intodisease progression about 1 year after the disease control, this groupis called IR (Intermediate Responder). Since most of this group isdetermined as SD about 9 weeks after the start of the treatment, “stablegroup” is also used interchangeably with IR (Intermediate Responder)group.

As used herein, “complete response group” refers to a group of subjectsdetermined to be complete response (CR, Complete response) when atherapeutic effect upon undergoing cancer therapy is determinedaccording to RECIST ver 1.1. A “complete response group” is also calleda CR group, which is interchangeably used herein. The present inventiondetects a case where a population of subjects comprises a completeresponse group (CR) in addition to a partial response group (PR) and acase where a population of subjects comprises a complete response group(CR) without comprising a partial response group (PR) as the same as apartial response group (PR).

As used herein, “response group” is used when a “partial response group”and “complete response group” are collectively called. This is alsocalled a “highly effective group”. In addition, a group where a longterm disease state control lasted for more than 1 year after startingthe treatment is called GR (Good responder). However, since most of thisgroup is identified as “partial response group” or “complete responsegroup” 9 weeks after starting treatment, “response group” can also beused interchangeably with GR (Good responder) group.

As used herein, “relative value” refers to a value obtained bycalculating a certain value while using another value as a baseline ofcomparison.

As used herein, the term “detection agent” broadly refers to all agentsthat are capable of detecting a substance of interest (e.g., cellsurface marker or the like).

As used herein, the “amount” of a certain cell subpopulation encompassesthe absolute number of certain cells and relative amount of a ratio in acell population.

As used herein, “threshold value” refers to a value that is determinedfor a certain variable value, where the value gives a certain meaningwhen the changing value is greater or less than the value. A thresholdvalue is also called a cut-off value herein.

As used herein, “ineffective group threshold value” refers to athreshold value used for identifying an ineffective group and stablegroup+response group in a given population of subjects. An ineffectivegroup threshold value is selected to achieve a predetermined sensitivityand specificity when selecting an ineffective group in a givenpopulation of subjects.

As used herein, “response group threshold value” refers to a thresholdvalue used for identifying a stable group and a response group in agiven population of subjects or in a given population of subjects fromwhich an ineffective group is removed using an ineffective groupthreshold value. A response threshold value is selected to achieve apredetermined sensitivity and specificity when selecting a responsegroup in a given population of subjects or in a given population ofsubjects from which an ineffective group is removed using an ineffectivegroup threshold value.

The term “about”, when used to qualify a numerical value herein, is usedto mean that the described value encompasses a range of values up to±10%.

As used herein, “flow cytometry” refers to a technique of measuring thenumber of cells, individual or other biological particles suspended in aliquid and individual physical/chemical/biological attributes.

(Cancer Immunotherapy)

Cancer immunotherapy is a method of treating cancer using a biologicaldefense mechanism of an organism. Cancer immunotherapy can be largelydivided into cancer immunotherapy from strengthening the immune functionagainst cancer and cancer immunotherapy from inhibiting the immuneevasion mechanism of cancer. Cancer immunotherapy further includesactive immunotherapy for activating the immune function in the body andpassive immunotherapy for returning immune cells with an immune functionactivated or expanded outside the body into the body. The biomarker ofthe present invention is understood to evaluate the overall balance ofthe CD4⁺ T-cell immunity to evaluate the overall tumor immunity itself,so that a therapeutic effect of all cancer immunotherapy can be broadlypredicted.

Examples of cancer immunotherapy include non-specificimmunopotentiators, cytokine therapy, cancer vaccine therapy, dendriticcell therapy, adoptive immunotherapy, non-specific lymphocyte therapy,cancer antigen specific T-cell therapy, antibody therapy, immunecheckpoint inhibition therapy and the like. The Examples of the presentspecification demonstrate that the biomarker of the present inventionaccurately predicts a therapeutic effect of especially, although notlimited to, immune checkpoint inhibition therapy.

PD-1 inhibitors are representative examples of immune checkpointinhibitors. Examples of PD-1 inhibitors include, but are not limited to,anti-PD-1 antibody nivolumab (sold as Opdivo™) and pembrolizumab. In onepreferred embodiment, nivolumab can be selected. Although not wishing tobe bound by any therapy, one of the reasons that therapy using nivolumabis preferred is because the Examples demonstrate that the use of thebiomarker of the present invention can clearly identify a responsivesubject and a non-responsive subject, and especially because it isrevealed that responsiveness and non-responsiveness can be clearlydistinguished by a specific threshold value. Of course, it is understoodthat the biomarker of the present invention can be utilized for otherPD-1 inhibitors to the same degree.

The present invention can also use PD-L1 inhibitors to the same extentas PD-1 inhibitors.

It is understood that anti-PD-1 antibodies achieve an anti-cancer effectby releasing the suppression of T-cell activation by a PD-1 signal. Itis understood that anti-PD-L1 antibodies also achieve an anticancereffect by releasing the suppression of T-cell activation by a PD-1signal. While the mechanism of PD-1 inhibiting a T-cell function is notfully elucidated, it is understood that an interaction between PD-1(programmed death 1) and PD-L1 or PD-L2 recruits a tyrosine phosphatase,SHP-1 or 2, to the cytoplasmic domain of PD-1 to inactivate a T-cellreceptor signaling protein ZAP70 to suppress activation of T-cells(Okazaki, T., Chikuma, S., Iwai, Y. et al.: A rheostat for immuneresponses: the unique properties of PD-1 and their advantages forclinical application. Nat. Immunol., 14, 1212-1218 (2013)). This isunderstood to be caused by the recruitment of SHP-1 or 2 to a partcalled an ITSM motif which dephosphorylates proximal signaling kinase ofa T-cell receptor in the vicinity. In other words, the memory of “beingstimulated by an antigen” is erased from a T-cell that has beenstimulated by an antigen.

PD-1 is expressed at a high level in killer T-cells and natural killercells, which have infiltrated a cancer tissue. It is understood that animmune response mediated by a PD-1 signal from PD-1 is attenuated byPD-L1 on tumor. While the immune response mediated by a PD-1 signal isattenuated by PD-L1, an effect of enhancing an anti-tumor immuneresponse is attained by inhibiting an interaction between PD-1 and PD-L1and/or signaling induced by an interaction with an anti-PD-1 antibody.

A PD-L1 inhibitor (e.g., anti-PD-L1 antibodies avelumab, durvalumab, andatezolizumab) is another example of an immune checkpoint inhibitor.

PD-L1 inhibitors bind and inhibit the aforementioned PD-1 pathway to thePD-L1 side to inhibit an interaction between PD-1 and PD-L1 and/orsignaling induced by an interaction to induce an anti-tumor immuneresponse. Although not wishing to be bound by any therapy, subjects whoare responsive or non-responsive to therapy that inhibits the PD-1pathway (e.g., anti-PD-1 antibody or anti-PD-L1 antibody) can be clearlyidentified by using the biomarker of the present invention in view ofthe results demonstrated in the Examples.

A CTLA-4 inhibitor (e.g., anti-CTLA-4 antibody ipilimumab ortremelimumab) is another example of an immune checkpoint inhibitor.

CTLA-4 inhibitors activate T-cells to induce an anti-tumor immuneresponse. T-cells are activated by an interaction of CD28 on the surfacewith CD80 or CD86. However, it is understood that surface expressedCTLA-4 (cytotoxic T-lymphocyte-associated antigen 4) preferentiallyinteracts with CD80 or CD86 with higher affinity than CD20 to suppressactivation, even for T-cells that have been activated. CTLA-4 inhibitorsinduce an anti-tumor immune response by inhibiting CTLA-4 to preventinhibition of an interaction between CD20 and CD80 or CD86.

In another embodiment, an immune checkpoint inhibitor may target animmune checkpoint protein such as TIM-3 (T-cell immunoglobulin and mucincontaining protein-3), LAG-3 (lymphocyte activation gene-3), B7-H3,B7-H4, B7-H5 (VISTA), or TIGIT (T cell immuno-receptor with Ig and ITIMdomain).

It is understood that the aforementioned immune checkpoints alsosuppress an immune response to autologous tissue, but immune checkpointsincrease in T-cells when an antigen such as a virus is present in vivofor an extended period of time. It is understood that tumor tissue isalso an antigen which is present in vivo for an extended period of time,so that an anti-tumor immune response is evaded by such immunecheckpoints. The aforementioned immune checkpoint inhibitors invalidatesuch an evasion function to achieve an anti-tumor effect. Although notwishing to be bound by any therapy, it is understood that the biomarkerof the present invention evaluates the balance of the overall anti-tumorimmune responses of humans so that it can be used as an indicator foraccurately predicting a therapeutic effect of such an immune checkpointinhibitor.

One embodiment of the present invention provides a compositioncomprising an immune checkpoint inhibitor. A composition comprising animmune checkpoint inhibitor can attain a significant therapeutic effectat a high probability by administration thereof to a subject who hasbeen selected by evaluation with the biomarker of the present invention.

The composition comprising an immune checkpoint inhibitor of the presentinvention is generally administered systemically or locally in an oralor parenteral form.

The dosage varies depending on the age, body weight, symptom,therapeutic effect, administration method, treatment time or the like,but is generally administered, for example, orally one to several timesa day in the range of 0.1 mg to 100 mg per dose per adult, or isadministered parenterally (preferably intravenously) one to severaltimes a day in the range of 0.01 mg to 30 mg per dose per adult orcontinuously administered intravenously in the range of 1 hour to 24hours per day. Of course, the dosage varies depending on variousconditions, so that an amount less than the above dosage may besufficient or an amount exceeding the range may be required.

For administration, a composition comprising an immune checkpointinhibitor can have a dosage form such as a solid agent or liquid agentfor oral administration or an injection, topical agent, or suppositoryfor parenteral administration. Examples of solid agents for oraladministration include tablets, pills, capsules, powder, granules andthe like. Capsules include hard and soft capsules.

The composition of the present invention includes one or more activeingredients (e.g., antibody to an immune checkpoint protein), which isdirectly used or is mixed with an excipient (lactose, mannitol, glucose,microcrystalline cellulose, starch, or the like), binding agent(hydroxypropyl cellulose, polyvinyl pyrrolidone, magnesiumaluminometasilicate, or the like), disintegrant (calcium celluloseglycolate or the like), lubricant (magnesium stearate or the like),stabilizer, solubilizing agent (glutamic acid, aspartic acid, or thelike), which is formulated in accordance with a conventional method foruse. The composition may also be coated with a coating agent (refinedsugar, gelatin, hydroxypropyl cellulose, hydroxypropyl methyl cellulosephthalate, or the like) or coated by two or more layers as needed.Capsules made of a substance that can be absorbed such as gelatin arealso encompassed.

The composition of the present invention comprises a pharmaceuticallyacceptable aqueous agent, suspension, emulsion, syrup, elixir or thelike when formulated as a liquid agent for oral administration. In sucha liquid agent, one or more active ingredients is dissolved, suspended,or emulsified in a commonly used diluent (purified water, ethanol, amixture thereof, or the like). Such a liquid agent may also contain ahumectant, suspending agent, emulsifier, sweetener, flavor, fragrance,preservative, buffer, or the like.

Examples of injections for parenteral administration include a solution,suspension, emulsion, and solid injection that is used by dissolving orsuspending it in a solvent at the time of use. An injection is used bydissolving, suspending or emulsifying one or more active ingredientsinto a solvent. Examples of solvents that are used include distilledwater for injections, saline, vegetable oil, propylene glycol,polyethylene glycol, alcohols such as ethanol, combination thereof, andthe like. Such an injection may also comprise a stabilizer, solubilizingagent (glutamic acid, aspartic acid, polysorbate or the like),suspending agent, emulsifier, analgesic, buffer, preservative, or thelike. They are prepared by sterilizing or aseptic operation in the finalstep. It is also possible to manufacture an aseptic solid agent such asa lyophilized product, which is dissolved in sterilized or asepticdistilled water for injection or another solvent before use.

(Cancer)

Examples of target cancer in the present invention include, but are notlimited to, melanoma (malignant melanoma), non-small cell lung cancer,renal cell cancer, malignant lymphoma (Hodgkin's or non-Hodgkin'slymphoma), head and neck cancer, urological cancer (bladder cancer,urothelial cancer, and prostate cancer), small cell lung cancer, thymiccarcinoma, gastric cancer, esophageal cancer, esophagogastric junctioncancer, liver cancer (hepatocellular carcinoma, intrahepaticcholangiocarcinoma), primary brain tumor (glioblastoma and primarycentral nervous system lymphoma), malignant pleural mesothelioma,gynecologic cancer (ovarian cancer, cervical cancer and uterine cancer),soft tissue sarcoma, cholangiocarcinoma, multiple myeloma, breastcancer, colon cancer and the like.

(Biomarker)

The present invention provides a novel biomarker for predicting atherapeutic effect of cancer immunotherapy. In one aspect, the T-cellcomposition of a subject is used as an indicator for predicting atherapeutic effect of cancer immunotherapy.

In one embodiment, a certain indicator of the T-cell composition of asubject at or greater than a response group threshold value indicatesthat the subject is a part of a response group to cancer immunotherapy.In another embodiment, a certain indicator of the T-cell composition ofa subject at or less than a response group threshold value indicatesthat the subject is a part of a response group to cancer immunotherapy.In yet another embodiment, a certain indicator of the T-cell compositionof a subject at or greater than an ineffective group threshold valueindicates that the subject is a part of an ineffective group to cancerimmunotherapy. In yet another embodiment, a certain indicator of theT-cell composition of a subject at or less than an ineffective groupthreshold value indicates that the subject is a part of an ineffectivegroup to cancer immunotherapy.

Those skilled in the art can determine a suitable threshold value foreach of such indicators. Those skilled in the art can predict a responseto cancer immunotherapy of a subject at the indicated sensitivity and/orspecificity by using the threshold value (ineffective group thresholdvalue and/or response group threshold value) disclosed herein.

For the indicators disclosed herein, those skilled in the art canappropriately determine a threshold value that would achieve a desiredsensitivity and specificity from results of determining an effect ofcancer immunotherapy of a reference subject group. The group of subjectswho are proven in the Examples of the present specification can beconsidered as a reference subject group. In other words, those skilledin the art can determine a threshold value from the results ofexperiments disclosed in the Examples or determine a new threshold valuefrom results of a reference subject population upon practicing thepresent invention.

Sensitivity refers to the ratio of the number of subjects with a givenfeature in selected targets to the total number of subjects with thegiven feature in a population of subjects when selecting subjects withthe given feature from among the population of subjects. For example,sensitivity is 100% when all subjects with a given feature in apopulation of subjects are selected. The sensitivity is 50% when half ofsubjects with a given feature in a population of subjects are selected.The sensitivity is 0% when none of the subjects with a given feature ina population of subjects are selected. Sensitivity is determined as, forexample, (number of subjects with a given feature among selectedtargets)/(total number of subjects with a given feature in a subjectpopulation). Determination with high sensitivity means that when it isdesirable to find a subject with a certain condition (e.g., ineffectivegroup with respect to cancer immunotherapy), such a subject is likely tobe definitively determined as being in such a condition.

The biomarker of the present invention which enables determination athigh sensitivity is very useful for ensuring the discovery of anineffective group with respect to a certain therapy. It is also possibleto select a threshold value so that sensitivity would be high inaccordance with such an objective.

Specificity refers to the ratio of the number of subjects with a givenfeature in selected targets to the total number of selected targets whenselecting a subject with the given feature from among a population ofsubjects. For example, specificity is 100% when all candidates selectedfrom among a subject population have the given feature. The specificityis 50% when half of the candidates selected from among a subjectpopulation have the given feature. The specificity is 0% when none ofthe candidates selected from among a subject population have the givenfeature. Specificity is determined as, for example, (number of subjectswith a given feature among selected targets)/(total number of selectedtargets). Determination with high specificity means that the probabilityof incorrectly determining a subject who is not in a certain condition(e.g., response group with respect to cancer immunotherapy) as not beingin such a condition (e.g., response group with respect to cancerimmunotherapy) is low.

The biomarker of the present invention which enables determination withhigh specificity is useful, for example for preventing a determinationthat would incorrectly determine a response group to a certain therapyas an ineffective group to discontinue therapy. It is also possible toselect a threshold value so that specificity would be high in accordancewith such an objective.

For example, when identifying a subject as a part of an ineffectivegroup with an indicator at or below a certain threshold value(ineffective group threshold value) when an increase of the indicator iscorrelated with the effect of cancer immunotherapy, subjects who aredetermined to be not a part of an ineffective group (i.e., stable groupor response group) despite being a part of an ineffective groupdecreases (sensitivity increases) for threshold values that are sethigher, but subjects who are determined as a part of an ineffectivegroup despite not being an ineffective group (e.g., stable group orresponse group) increases (decrease in specificity). In contrast,subjects who are determined as a part of an ineffective group despitenot being a part of an ineffective group (i.e., stable group or responsegroup) decreases (specificity increases) for threshold values that areset lower, but subjects who are determined as not a part of anineffective group (i.e., stable group or response group) despite being apart of an ineffective group increases (sensitivity decreases).

For the biomarker of the present invention, a threshold value can be setand used so that specificity and/or sensitivity is very high, so thatthe biomarker of the present invention can be used as an unprecedentedand advantageous marker for predicting a therapeutic effect of cancerimmunotherapy. Those skilled in the art can also suitably set athreshold value in accordance with the objective in such a range ofthreshold values at which both the specificity and sensitivity are veryhigh. It should be understood that a proximate value of a specific valuecan be used as long as determination of interest can be performed evenwhen a specific value is shown as an example of a threshold value.

The ratio of CD62L^(low) T-cells in CD4⁺ T-cells of a subject can beused as an indicator for predicting a response to cancer immunotherapyof the subject, e.g., as an indicator for selecting an ineffectivegroup. The inventors have discovered that the ratio of CD62L^(low)T-cells in CD4⁺ T-cells higher than an ineffective group threshold valuecan predict at a very high precision that the subject is not a part ofan ineffective group to cancer immunotherapy in such a case.

An ineffective group threshold value for the ratio of CD62L^(low)T-cells in CD4⁺ T-cells can be appropriately determined by those skilledin the art based on a reference, or a threshold value (Cutoff) shown inFIG. 6 can be used as an ineffective group threshold value. It should benoted that the ratio may be denoted hereinafter as percent (%).

For example, when using 19.4 as an ineffective group threshold value inthe results of FIG. 6, it is understood that the ratio of CD62L^(low)T-cells in CD4⁺ T-cells can be used as a biomarker for determiningwhether a subject is a part of an ineffective group with sensitivity of92.9% and specificity of 96.7%.

When similarly using a value of 14.45 or less (e.g., 14.45, 13.8, 13.3,12.3, or 10.9) as an ineffective group threshold value for the ratio ofCD62L^(low) T-cells in CD4⁺ T-cells in the results of FIG. 6, anineffective group can be predicted with the ratio as a biomarker with100% specificity.

When using a value of 22.55 or greater (e.g., 23.1, 24.1, 24.8, 25.05,25.45, 25.95, 27, 28.75, or the like) as an ineffective group thresholdvalue for the ratio of CD62L^(low) T-cells in CD4⁺ T-cells, anineffective group can be predicted with the ratio as a biomarker with100% sensitivity.

In other words, an ineffective group threshold value as a ratio ofCD62L^(low) T-cells in CD4⁺ T-cells can be in the range of about 10 toabout 30(%). Examples of such an ineffective group threshold valueinclude about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, and 30(%).

The ratio of Foxp3⁺CD25⁺ T-cells in CD4⁺ T-cells of a subject can alsobe used as an indicator for predicting a response to cancerimmunotherapy of the subject, such as an ineffective group thresholdvalue. The inventors have discovered that a ratio of Foxp3⁺CD25⁺ T-cellsin CD4⁺ T-cells in a subject derived sample lower than an ineffectivegroup threshold value indicates that the subject is not a part of anineffective group with respect to cancer immunotherapy.

The ineffective group threshold value for a ratio of Foxp3⁺CD25⁺ T-cellsin CD4⁺ T-cells of a subject can be appropriately determined by thoseskilled in the art from a reference subject. Such an ineffective groupthreshold value can be in a range of about 2 to about 4(%). Examples ofthreshold values include about 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0(%).

Indicators of the T-cell composition disclosed herein can be used incombination when preferred. Since the inventors have discovered thatmultiple indicators independently exhibit correlation withresponsiveness, it is understood that multiple indicators, which arecombined and used as an indicator for responsiveness, can furtherimprove the precision of prediction.

When two or more indicators are combined as an indicator ofresponsiveness, an indicator represented by a formula using any numberof variables can be used. When multiple indicators (X₁, X₂, X₃ . . .X_(n)) are used, examples of indicators of responsiveness include butare not limited to the following:

F=a ₁ X ₁ ^(b1) +a ₂ X ₂ ^(b2) +a ₃ X ₃ ^(b3) . . . +a _(n) X _(n) ^(bn)

F=X ₁ ^(c1) *X ₂ ^(c2) *X ₃ ^(c3) . . . *X _(n) ^(cn)

wherein each of a, b, and c is any real number. Responsiveness can bepredicted from the magnitude of the indicator that is calculated by sucha formula. Multivariate analysis by logistic regression or discriminantanalysis can be performed on the novel indicators discovered by theinventors to determine a coefficient for use as an indicator ofresponsiveness to cancer immunotherapy of a subject.

While indicators that are combined are not limited, the inventors havediscovered indicators such as the amount of a CD4⁺ T-cell subpopulationcorrelated with a dendritic cell stimulation in an anti-tumor immuneresponse, amount of a dendritic cell subpopulation correlated with adendritic cell stimulation in an anti-tumor immune response, the amountof a CD8⁺ T-cell subpopulation correlated with a dendritic cellstimulation in an anti-tumor immune response, amount of regulatoryT-cells or a CD4⁺ T-cell subpopulation correlated with regulatoryT-cells, and amount of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation.Indicators that predict responsiveness by different mechanisms can becombined for use as an indicator exhibiting stronger correlation withresponsiveness, which is not false correlation.

For example, two or more indicator, such as three, four, five, or moreindicators, that are selected from the group consisting of:

an amount of a CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a CCR7⁻ CD4⁺ T-cell subpopulation;an amount of a LAG-3⁺CD62L^(low)CD4⁺ T-cell subpopulation;an amount of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a CD45RA⁻CD4⁺ T-cell subpopulation;an amount of a CD45RO⁺CD4⁺ T-cell subpopulation;an amount of an HLA-DR⁺ dendritic cell subpopulation;an amount of a CD80⁺ dendritic cell subpopulation;an amount of a CD86⁺ dendritic cell subpopulation;an amount of a PD-L1⁺ dendritic cell subpopulation;an amount of a CD62L^(low)CD8⁺ T-cell subpopulation;an amount of a CD137⁺CD8⁺ T-cell subpopulation;an amount of a CD28⁺CD62L^(low)CD8⁺ T-cell subpopulation;an amount of a CCR4⁺CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD127⁺CD25⁺CD4⁺ T-cell subpopulation;an amount of CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation; andan amount of CD4⁺Foxp3⁺CD25⁺ T-cell subpopulation;can be used in combination.

It is possible to show that a subject is not a part of an ineffectivegroup with respect to cancer immunotherapy by combining indicators, asresponsiveness to cancer immunotherapy. Similarly, it is possible todetermine that a subject is a part of a response group with respect toresponse to cancer immunotherapy by combining indicators, asresponsiveness to cancer immunotherapy.

Typically, responsiveness can be predicted by formula F(X, Y) using twoindicators (X, Y) disclosed herein as variables. In some cases, aformula is a relative value of X to Y.

Any function of X and Y (F(X, Y)) can be used as a relative value of Xto Y. Especially when it is understood that X is positively correlatedwith responsiveness and Y is negatively correlated with responsiveness,any function of X and Y (F(X, Y)), which monotonically increases withrespect to X and monotonically decreases with respect to Y, can be used,but the formula is not limited thereto. With two or more variablesrepresenting responsiveness, a formula representing responsiveness canbe found by regression from logistic regression or the like bycalculating the contribution of each variable to responsiveness.

Examples of F(X, Y) representing responsiveness include, but are notlimited to the following.

F=aX ^(r) +bY ^(s)

F=X ^(r) *Y ^(s)

wherein a, b, r, and s are any real numbers.

Integers can be used as r and s for simplicity of the formula. In someembodiments, examples of relative values of X to Y include, but are notlimited to, X^(n)/Y^(m) (n and m are any integer) such as X/Y and X²/Y.

When each of factors X and Y indicates responsiveness to therapy fromdifferent mechanisms, combination of such indicators can make theprediction of responsiveness more accurate. The study by the inventorsshows that a formula with r and s in the range of −5 to 5 can be used toaccurately predict responsiveness to cancer immunotherapy of a subject.

In one embodiment, a subject can be shown to be not a part of anineffective group with respect to cancer immunotherapy by using theamount of T-cells correlated with a dendritic cell stimulation in ananti-tumor immune response as X and the amount of regulatory T-cells ora CD4⁺ T-cell subpopulation correlated with regulatory T-cells as Y. Inthis case, it is demonstrated that responsiveness to cancerimmunotherapy of a subject can be accurately predicted using a formulawith r and s in the range of −5 to 5. Examples of such a formula includeX/Y, X²/Y, X³/Y, X⁴/Y, X⁵/Y, X/Y², X²/Y², X³/Y², X⁴/Y², X⁵/Y², X/Y³,X²/Y³, X³/Y³, X⁴/Y³, X⁵/Y³, X/Y⁴, X²/Y⁴, X³/Y⁴, X⁴Y⁴, X⁵/Y⁴, X/Y⁵,X²/Y⁵, X³/Y⁵, X⁴/Y⁵, X⁵/Y⁵, and the like.

Examples of the present specification show that F=X^(2.475)/Y can beused as an indicator by logistic regression for a combination of theamount of CD62L^(low) T-cells in CD4⁺ T-cells of a subject (X) and theamount of Foxp3⁺CD25⁺ T-cells in CD4⁺ T-cells (Y), but those skilled inthe art can appropriately derive a different combination or a differentformula for the indicators disclosed herein by a similar analysis.

In regression analysis, a result from a sample greater than the numberof combined variables +1 can be used to calculate a coefficient in aformula of a combination of variables. When a form of formula in acombination of two indicators is found by regression analysis,regression analysis is performed using a result in at least foursamples. Preferably, regression analysis is performed using results in20 or more samples. More preferably, regression analysis is performedusing results in 30 or more samples. Regression analysis with a greaternumber of samples can be advantageous in that a combination ofindicators that predicts responsiveness of a subject more accurately canbe found.

In one embodiment, the amount of CD62L^(low) T-cells in CD4⁺ T-cells ofa subject (X) and the amount of Foxp3⁺CD25⁺ T-cells in CD4⁺ T-cells (Y)can be used as an ineffective threshold value as a combined indicator.For example, a relative value of X to Y can be used as an indicator forpredicting a response with respect to cancer immunotherapy of a subject.

For example, the present invention can calculate variables (X, Y), witha value selected from the group consisting of

an amount of a CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a CCR7⁻CD4⁺ T-cell subpopulation;an amount of a LAG-3⁺CD62L^(low)CD4⁺ T-cell subpopulation;an amount of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a CD45RA⁻CD4⁺ T-cell subpopulation;an amount of a CD45RO⁺CD4⁺ T-cell subpopulation;an amount of an HLA-DR⁺ dendritic cell subpopulation;an amount of a CD80⁺ dendritic cell subpopulation;an amount of a CD86⁺ dendritic cell subpopulation;an amount of a PD-L1⁺ dendritic cell subpopulation;an amount of a CD62L^(low)CD8⁺ T-cell subpopulation;an amount of a CD137⁺CD8⁺ T-cell subpopulation; andan amount of a CD28⁺CD62L^(low)CD8⁺ T-cell subpopulation;

as (X).

The method of the present invention can also calculate variables (X, Y),with the amount of a regulatory T cell subpopulation or a CD4⁺ T cellsubpopulation correlated with regulatory T cells as (Y). The method ofthe present invention can also calculate variables (X, Y), with a valueselected from the group consisting of:

an amount of CCR4⁺CD25⁺CD4⁺ T-cell subpopulation;an amount of CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation;an amount of CD127⁺CD25⁺CD4⁺ T-cell subpopulation;an amount of CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation; andan amount of CD4⁺Foxp3⁺CD25⁺ T-cell subpopulation;

as (Y).

The present invention further provides a method of identifying aresponse group (PR) and stable group (SD) in a subject populationdetermined not to be a part of an ineffective group using the above (X,Y). A method of identifying the response group (PR) and stable group(SD) can predict whether a subject is a part of the response group (PR)or stable group (SD) by calculate variables (Z, W), with

an amount of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulationas (Z) and a value selected from the group consisting ofan amount of a CD4⁺CD25⁺ T-cell subpopulation;an amount of a CD4⁺Foxp3⁺ T-cell subpopulation;an amount of a CD4⁺Foxp3⁺CD25⁺ T-cell subpopulation;an amount of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation;an amount of a CCR4⁺CD25⁺CD4⁺ T-cell subpopulation; andan amount of a CD127⁺CD25⁺CD4⁺ T-cell subpopulation;

as (W).

The relative value of X to Y is not particularly limited, but anyfunction of X and Y (F(X, Y)), which monotonically increases withrespect to X and monotonically decreases with respect to Y, can be used.For example, such a function can be

F(X,Y)=G(X)/H(Y); or

F(X,Y)=G(X)−H(Y)

wherein G(X) and H(Y) can be monotonically increasing functions withrespect to X and Y, respectively. For example, G(X) can be X^(R),log_(R)X, R^(X), or the like, wherein R is any real number satisfyingthe condition and is preferably a positive integer. For example, H(Y)can be Y^(R), log_(R)Y, or R^(Y), or the like, wherein R is any realnumber satisfying the condition and is preferably a positive integer. Insuch a form, the accuracy of prediction can be improved by using apositive prediction for a therapeutic effect of cancer immunotherapy ofX in combination with a negative prediction for cancer immunotherapy ofY as an indicator.

Examples of relative values of X to Y include, but are not limited to,X^(n)/Y^(m) (n and m are any positive real numbers) such as X/Y andX²/Y. When each of factors X and Y indicates responsiveness to therapyfrom different mechanisms, combination of such indicators can make theprediction of responsiveness more accurate.

A function using Z and W is not particularly limited. Any function of Zand W (J(Z, W)) can be used. Examples of such a function can be

J(Z,W)=K(Z)*L(W); or

J(Z,W)=K(Z)+L(W)

wherein K(Z) and L(W) can typically be functions which monotonicallyincrease with respect to Z and W, respectively. For example, K(Z) can beZ^(R), log_(R)Z, R^(Z), or the like, wherein R is any real numbersatisfying the condition and is preferably a positive integer. Forexample, L(W) can be W^(R), log_(R)W, R^(W), or the like, wherein R isany real number satisfying the condition and is preferably a positiveinteger. Based on J(Z, W), accuracy of determination of a response group(PR) and stable group (SD) in an ineffective group can be improved.Examples of relative values of Z to W include, but are not limited to,Z^(n)*W^(m) (wherein n and m are any real number), such as W⁵*Z. Wheneach of factors Z and W indicates responsiveness to therapy fromdifferent mechanisms, combination of such indicators can make theprediction of responsiveness more accurate.

When the amount of CD62L^(low) T-cells in CD4⁺ T-cells is X and theamount of Foxp3⁺CD25⁺ T-cells in CD4⁺ T-cells is Y, X/Y can be used asan indicator for predicting a response to cancer immunotherapy of asubject. The inventors have discovered that a subject with a high X/Y isshown to be not a part of an ineffective group with respect to cancerimmunotherapy. Thus, a value of X/Y can be used as an ineffective groupthreshold value.

An ineffective group threshold value for X/Y can be appropriatelydetermined by those skilled in the art based on a reference, or a value(Cutoff) shown in FIG. 8 can be used as an ineffective group thresholdvalue.

When 7.35 is used as an ineffective group threshold value of X/Y, anineffective group can be predicted using X/Y as a biomarker fordetermining whether a subject is a part of an ineffective group withsensitivity of 71.4% and specificity of 100%.

When a value of 7.35 or less (e.g., 7.35, 6.83, 6.31, 5.64, 5.01, or thelike) is used as an ineffective group threshold value of X/Y, this canpredict an ineffective group as a biomarker with specificity of 100%.

When a value of 9.305 or greater (e.g., 9.895, 10.19, 11.71, 12.07,12.32, 12.42, or the like) is used as an ineffective group thresholdvalue of X/Y, this can predict an ineffective group as a biomarker withsensitivity of 100%.

In other words, an ineffective group threshold value of X/Y can be inthe range of about 5 to about 13. Examples of an ineffective groupthreshold value of X/Y include about 5, 6, 7, 8, 9, 10, 11, 12, and 13.

Furthermore, as a relative value of X to Y, X²/Y can be used as anineffective group threshold value, which is an indicator for predictinga response to cancer immunotherapy of a subject. The inventors havediscovered that a subject with high X²/Y is shown to be highly unlikelya part of an ineffective group with respect to cancer immunotherapy.

An ineffective group threshold value for X²/Y can be appropriatelydetermined by those skilled in the art based on a reference, or athreshold value (Cutoff) shown in FIG. 10 can be used as an ineffectivegroup threshold value.

When 174.3 is used as an ineffective group threshold value for X²/Y,X²/Y can predict an ineffective group as a biomarker for determiningwhether a subject is a part of an ineffective group with sensitivity andspecificity both at 100%.

110.6, 118.2, 134.9, 151.6, 157.4, 174.3, 194.2, 202.3, 208.3, and thelike can be used as other values of ineffective group threshold valuesfor X²/Y.

In other words, an ineffective group threshold value for X²/Y can be inthe range of about 110 to about 210. Examples of ineffective groupthreshold values for X²/Y include about 110, 120, 130, 140, 150, 160,170, 180, 190, 200, and 210.

Those skilled in the art can use other relative values of X to Y bysetting an appropriate threshold value at least from the data disclosedherein.

It is also possible to distinguish PR and SD (show that a subject ispart of a response group) using results of calculating (e.g.,multiplying) two or more indicators (biomarkers, BM) of the presentinvention. In one embodiment, a value of Z^(n)*W^(m) (n and m arepositive real numbers), with a first biomarker as “Z” and a secondbiomarker as “W”, can be used to distinguish PR and SD, but this is notlimited thereto. It is also possible to use results from calculating(e.g., adding and/or multiplying) three or more biomarkers fordistinguishing PR and SD. For example, when a PR group and an SD groupare distinguished using the product W*Z of the ratio of CD25⁺Foxp3⁺CD4⁺T-cells in CD4⁺ T-cells (W) and the ratio of ICOS⁺CD62L^(low)CD4⁺T-cells in CD62L^(low)CD4⁺ T-cells (Z), it was demonstrated that athreshold value of W*Z of 1.816 can be used as a biomarker withsensitivity of 80% and specificity of 89.5% (bottom figure in the middleof FIG. 21). Alternatively, it was demonstrated that Z*W⁵ can be used asa biomarker with sensitivity of 54.55% and specificity of 100%.

In addition, any function of Z and W (J(Z, W)) can be used as disclosedabove. For example, a formula such as J=Z^(r)*W^(s) with r and s in therange of −5 to 6 can be used to accurately predict the responsiveness tocancer immunotherapy of a subject. Examples of such a formula includeZ*W, Z²*W, Z³*W, Z⁴*W, Z⁵*W, Z⁶*W, Z*W², Z²*W², Z³*W², Z⁴*W², Z⁵*W²,Z⁶*W², Z*W³, Z²*W³, Z³*W³, Z⁴*W³, Z⁵*W³, Z⁶*W³, Z*W⁴, Z²*W⁴, Z³*W⁴,Z⁴*W⁴, Z⁵*W⁴, Z⁶*W⁴, Z*W⁵, Z²*W⁵, Z³*W⁵, Z⁴*W⁵, Z⁵*W⁵, Z⁶*W⁵, Z*W⁶,Z²*W⁶, Z³*W⁶, Z⁴*W⁶, Z⁵*W⁶, Z⁶*W⁶, and the like. Examples in the presentspecification show that Z*W⁵ can be used as a preferred predictionformula, which combines the ratio of CD25⁺Foxp3⁺CD4⁺ T-cells in CD4⁺T-cells (W) and the ratio of ICOS⁺CD62L^(low)CD4⁺ T-cells inCD62L^(low)CD4⁺ T-cells (Z) by logistic regression or the like (FIGS. 25and 26). Meanwhile, those skilled in the art can appropriately derive adifferent combination or different formula for indicators disclosedherein by a similar analysis.

The present specification further provides an indicator that can be usedto distinguish a response group (complete response+partial response) andstable group (intermediate group) among a subject population determinedas not part of an ineffective group.

The ratio of Foxp3⁺CD25⁺ T-cells in CD4⁺ T-cells can be used as anindicator for predicting a response to cancer immunotherapy of a subjectwho has been predicted as not a part of an ineffective group. Theinventors have discovered that a high ratio of Foxp3⁺CD25⁺ T-cells inCD4⁺ T-cells in subjects shown as not a part of an ineffective groupmeans that the subject is highly likely to be a part of a response groupwith respect to cancer immunotherapy. CD4⁺Foxp3⁺CD25⁺ T-cells areregulatory T-cells with an immunosuppressive property, so that it wasunexpected to find that a subject with a high ratio of such cells ishighly likely to respond to cancer immunotherapy.

A ratio of LAG-3⁺CD62L^(low)CD4⁺ T cell subpopulation in CD62L^(low)CD4⁺T-cells, or a ratio of PD-1⁺CD62L^(low)CD4⁺ T cell subpopulation inCD62L^(low)CD4⁺ T-cells can be used as an indicator for predicting aresponse to cancer immunotherapy of a subject who has been predicted asnot a part of an ineffective group. The inventors have discovered thatthese cell subpopulations can be used to distinguish a response group(complete response+partial response) and stable group (intermediategroup).

The method of the present invention can use be used, for example, acomparison of a relative value of X to Y with a threshold value(ineffective group threshold value) comprising measuring the amount ofCD80⁺ dendritic cells (X) and measuring the amount of aCD28⁺CD62L^(low)CD8⁺ T-cell (Y) as an indicator for predicting that thesubject is not a part of an ineffective group with respect to the cancerimmunotherapy.

It is possible to predict that a subject is not a part of an ineffectivegroup with any biomarker disclosed herein combined with any ineffectivegroup threshold value. In addition, it is possible to predict anineffective group using a threshold value determined for such indicatorsas an ineffective group threshold value, and use a threshold value forthe ratio of Foxp3⁺CD25⁺ T-cells in CD4⁺ T-cells for predicting that asubject population (preferably a subject population with an ineffectivegroup excluded) is a part of a response group with respect to cancerimmunotherapy as a response group threshold value.

Alternatively, a method of identifying a response group (PR) and astable group (SD) in a subject population determined as not a part of anineffective group is provided. A method of identifying a response group(PR) and a stable group (SD) can predict whether a subject is a part ofa response group (PR) or a stable group (SD) by calculating variables(Z, W), with

an amount of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulationas (Z) and a value selected from the group consisting of:an amount of a CD4⁺CD25⁺ T-cell subpopulation;an amount of a CD4⁺Foxp3⁺ T-cell subpopulation;an amount of a CD4⁺Foxp3⁺CD25⁺ T-cell subpopulation;an amount of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation;an amount of a CCR4⁺CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD127⁺CD25⁺CD4⁺ T-cell subpopulation;

as (W).

A response group threshold value for the ratio of Foxp3⁺CD25⁺ T-cells inCD4⁺ T-cells can be appropriately determined by those skilled in the artbased on a reference, or a result shown in FIG. 12 can be appropriatelyselected as a response group threshold value. It should be noted thatthe ratio may be denoted hereinafter as percent (%).

When 2.05 is used as a response group threshold value for the ratio ofFoxp3⁺CD25⁺ T-cells in CD4⁺ T-cells, the ratio of Foxp3⁺CD25⁺ T-cells inCD4⁺ T-cells can be used as a biomarker for predicting whether a subjectis a part of a response group with sensitivity of 52.6% and specificityof 100%.

When a value of 2.05 or less (e.g., 2.05, 1.895, 1.76, 1.7, 1.61, or thelike) is used as a response group threshold value for the ratio ofFoxp3⁺CD25⁺ T-cells in CD4⁺ T-cells, this can be used for predicting aresponse group as a biomarker with specificity of 100%.

When a value of 3.35 or greater (e.g., 3.35, 3.63, 4.365, or the like)is used as a response group threshold value for the ratio of Foxp3⁺CD25⁺T-cells in CD4⁺ T-cells, this can be used for predicting a responsegroup as a biomarker with sensitivity of 100%.

In other words, a response group threshold value for the ratio ofFoxp3⁺CD25⁺ T-cells in CD4⁺ T-cells can be in the range of about 1.6 toabout 4.4(%). Examples of a response group threshold value for the ratioof Foxp3⁺CD25⁺ T-cells in CD4⁺ T-cells include about 1.6, 1.7, 1.8, 1.9,2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3,3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, and 4.4(%).

Another aspect of the present invention provides a method of applyingcancer immunotherapy to a subject selected with any of the biomarkersdisclosed above (preferably a combination of biomarkers). One embodimentprovides a method of administering an immune checkpoint inhibitor to asubject in which any one of the above biomarkers is in a state indicatedby any one of the threshold values disclosed herein.

(Fractionation/Separation of Cells)

A sample for fractionation/separation of T-cells can be suitablycollected from a subject using a conventional method. For example, thiscan be collected from peripheral blood, bone marrow, tumor tissue,hematopoietic tissue, spleen, normal tissue, lymph, or the like of asubject. Sample collection from peripheral blood can be advantageous forbeing simple and non-invasive.

The composition of T-cells in a sample of a subject can be measured bythose skilled in the art using a conventional method. Generally, thenumber of cells that are positive for a marker (e.g., CD4) defining acell subpopulation of interest in a sample can be measured using flowcytometry or the like. Some embodiments of the present inventioncomprise measuring the amount of CD62L^(low) T-cells in CD4⁺ T-cells (X)and/or the amount of FoxP3⁺CD25⁺ T-cells in CD4⁺ T-cells (Y). Themeasurement of the composition of a cell population generally uses flowcytometry, but may use a method using an antibody array orimmunostaining on a sample comprising cells, protein expression analysisin a sample comprising cells (e.g., western blot, mass spectrometry,HPLC, or the like), mRNA expression analysis in a sample comprisingcells (microarray or next generation sequencing), or the like.

To measure the cell count in each cell subpopulation such asCD62L^(low)CD4⁺ T-cell subpopulation and CD4⁺CD25⁺CD4⁺Foxp3⁺CD25⁺ T-cellsubpopulation, the measurement may be found by experimentally removingcells other than subpopulations of each kind from all cells. There is akit for the materialization thereof. For example, cells corresponding toa CD4⁺CD62L^(low) T-cell subpopulation can be separated from peripheralblood without using a CD4 antibody or CD62L antibody when a CD4⁺EffectorMemory T cell isolation kit, human (Militenyi Biotech) is used. This isachieved by counting and recording the total live cell count, andcounting and recording the number of cells obtained using this kit. Whena CD4⁺CD25⁺Regulatory T-cell isolation kit, human (Militenyi Biotech) isused, the cell count corresponding to a CD4⁺CD25⁺CD4⁺Foxp3⁺CD25⁺ T-cellsubpopulation can be found without using an anti-FoxP3 antibody. SinceFoxP3 is localized in the nucleus in cells, this has an advantage ofeliminating a step for staining a molecule in the nucleus. As a similarkit, CD4⁺CD25⁺CD127^(dim/−)Regulatory T cell isolation kit, human(Militenyi Biotech) or CD25⁺CD49d⁻ Regulatory T cell isolation kit,human (Militenyi Biotech) can also be selected.

An antibody does not need to be used. Antibodies that can specificallyrecognize and bind a molecule expressed on individual cells are preparedso that they can emit color when bound to a molecule expressed on thecell surface or inside the cells. The antibodies are then detected tomeasure the number of cells that are emitting color. Since the moleculesexpressed on the cell surface or inside the cells are proteins, mRNAencoding a protein when the protein is expressed is also formed in thecells. In other words, it is sufficient to examine mRNA in individualcells to examine the presence/absence of mRNA encoding a protein ofinterest. This is made possible by a single cell gene expressionanalysis, i.e., mRNA analysis at a single cell level. Examples of singlecell gene expression analysis include 1) a method of next generationsequencing using Quartz-Seq, 2) a method of isolating cells using aFluidigm Cl System or ICELL8 Single-Cell System to isolate cells andprepare a library with SMART-Seq v4, 3) a method of separating cellswith a cell sorter and measuring the cells with quantitative PCR usingan Ambion Single Cell-to-CT kit, 4) CyTOF SYSTEM (Helios), and the like.

In other words, blood is obtained, live cells are counted, and cells areseparated with a cell sorter or the like. For example, Ambion SingleCell-to-CT kit can be used on the individual separated cells to measurethe expression level of a specific gene with an apparatus forquantitative PCR. Based on the result, individual cells are examined asto which subpopulation among CD62L^(low) CD4⁺ T-cell subpopulation andCD4⁺Foxp3⁺CD25⁺ T-cell subpopulation the cells fall under, and thenumber of cells falling under each subpopulation is counted. The ratioof the numbers (ratio of x to y) is then found. Examples of candidategenes whose expression is examined include αβTCR, CD3, CD4, CD25, CTLA4,GITR, FoxP3, STATS, FoxO1, FoxO3, IL-10, TGFbeta, IL-35, SMAD2, SMAD3,SMAD4, CD62Llow, CD44, IL-7R (CD127), IL-15R, CCR7low, BLIMP1, and thelike.

As shown in the Example of the present specification, the genes whoseexpressions are increased in CD62L^(low)CD4⁺ T-cells compared toCD62L^(high)CD4⁺ T-cells include: AURAKA, CCL17, CD101, CD24, FOXF1,GZMA, GZMH, IL18RAP, IL21, IL5RA, ND2, SMAD5, SMAD7, and VEGFA (FIG. 34a). An amount and/or ratio of a cell subpopulation can be determined bymeasuring the expression of these genes, thereby determining which Tcell subpopulation the obtained T cells belong to.

As shown in the Example of the present specification, the genes whoseexpressions are increased in CD62L^(high)CD4⁺ T-cells compared toCD62L^(low)CD4⁺ T-cells include: BACH2, CCL28, CCR7, CD27, CD28, CD62L,CSNK1D, FOXP1, FOXP3, IGF1R, IL16, IL27RA, IL6R, LEF1, MAL, and TCF7(FIG. 34a ). An amount and/or ratio of a cell subpopulation can bedetermined by measuring expression of these genes, thereby determiningwhich T cell subpopulation the obtained T cells belong to.

Measurement of the ratio of cell subpopulations or comparison with athreshold value in the present invention may use a reference sample witha defined signal. Signals between a reference prepared to induce afluorescent signal corresponding to a given cell subpopulation (e.g.,particle to which a fluorescent pigment is attached) and a samplecomprising a cell population can be compared to measure the amount andratio of a cell population in the sample by comparison with a reference.Signals between a reference prepared to induce a fluorescent signalcorresponding to a predetermined threshold value (e.g., particle towhich a fluorescent pigment is attached) and a sample comprising a cellpopulation can also be compared to determine the presence/absence or themarker of the present invention in the T-cell composition in the sampleby a comparison with a reference.

When a specific marker is determined to be high (high expression) or low(low expression) in the present invention, those skilled in the art canuse a classification baseline for expression intensity that is commonlyused in the art. For example, it is possible to divide CD62L intoCD62L^(low) and CD62L^(high) using the signal intensity corresponding toa 10E2 signal when using a PE-labeled anti-human CD62L antibody as theboundary.

In one embodiment, CD62L can be determined as high (high expression) orlow (low expression) as follows. An antibody which is used as a negativecontrol of the same isotype of an anti-CD62L antibody is prepared. Theantibody used as a negative control should not recognize (bind) anyantigen on a T-cell, but may non-specifically adsorb thereto. Forexample, an antibody sold as an isotype control is used. The samefluorescent label is used for an anti-CD62L antibody and the negativecontrol. After preparation, respective fluorescence patterns areoverlaid. In a typical pattern, the isotype control has a peak at aportion with a low level of fluorescence while the anti-CD62L antibodyhas a peak where the fluorescence level is high, and the fluorescencelevel slowly decreases where the fluorescence is lower (FIG. 28). InFIG. 28, the purple line is the staining pattern of the negative controland the area under the line which is colored with light blue is thestaining pattern of the anti-CD62L antibody. The two patterns arecompared. While some areas have the same fluorescence level as thenegative control, it is determined that the entire peak of the negativecontrol has shifted to the right (=where it is stained). Generally, itis determined thereby that almost all cells were stained by an antibody.

The determination of a boundary of low and high on the x axis (FL4-H) isnow discussed. The right side of the figure is a schematic diagram whenassuming that the peaks of low and high are divided. The peak of highappears to be horizontally symmetric, but low has a composite peak thatcannot be considered horizontally symmetric. The peak of high is locatedat where FL4-H is, at about 400. The maximum amount of FL4-H of the peakof high (=A) is about 2,000. If the peak of high is consideredhorizontally symmetric, the inherent minimum amount of high (=B), whichis separated by the same distance from FL4-H with the peak to A and ison the opposite side of the peak from A, is about 90. Up to this area,there should be an overlap with the peak of low. While the peak of highhorizontally symmetric, horizontal symmetry is lost near D. In otherwords, D can be inferred to be the inherent maximum value of the peak oflow, which means that there is a peak of low up to near D. Ultimately,high and low can be divided at the center of D, which is the maximumvalue of low, and B, which is the minimum value of high, i.e., C. Thisvalue corresponds to 10E2. In other words, the range of high can be C toA, and the range of low can be E to C. The area formed by the peak andeach range corresponds to the cell count. The position of C on BD shouldvary depending on the ratio of sizes of peaks of high and low, sharpnessof peaks or the like, but the difference from cases where the positionof C is at the center of BD is considered small.

FIG. 29 (FIGS. 29A-29B) shows histograms for CD62L according to FACSanalysis. It is understood that CD62L^(low) can be separated veryclearly with 10E2 as the boundary.

As used herein, “flow cytometry” refers to a technique of measuring thenumber of cells, individual or other biological particles suspended in aliquid and individual physical/chemical/biological attributes.

Various cells are analyzed using a flow cytometry technique. Inparticular, differentiation of blood cells can be determined using aflow cytometry technique. Such a determination of differentiation isstarting to be used in diagnosis in addition to research.

Examples of advantages of flow cytometry include: the ratio accountedfor blast cells can be readily found; specificity and sensitivity arehigh; it is highly reproducible; a large number of cells can beanalyzed; the time required is short; and the like.

An apparatus using this technique is referred to as a “flow cytometer”.A flow cytometer is an equipment for measuring optical characteristicsof a suspended matter (cell) from a homogeneous suspension of cells.Cells pass through the focal point of a laser beam on a liquid flow.When passing, optical characteristics of forward scatter, side scatter,and one or more fluorescent light with different wavelengths can besimultaneously measured for individual cells at 500 to 4000 cells persecond to quickly and accurately measure biological characteristics suchas the cell size, internal structure, the amount of nucleic acid orvarious antigens that are present in the cell membrane, cytoplasm, orthe nucleus.

Scattered light is light scattered to the surrounding after a laser hitsa cell. Forward scatter (FSC) is detected in front with respect to thelaser optical axis. The scatter light intensity is proportional to thesurface area of a cell. In other words, it is understood that a cell islarge if the FSC value is relatively large, and the cell is small if theFSC value is small. Side scatter (SSC) is detected at a position that is90° (perpendicular) to the laser optical axis. The scatter lightintensity is proportional to the state of intracellular structure orcell granule. In other words, it is understood that the internalstructure of a cell is complex if the SSC value is relatively large, andthe internal structure of a cell is simple if the SSC value is small.

Results of flow cytometry can be typically expressed as a dot plot, withFSC as the X axis and SSC as the Y axis. Each cell is represented by asingle dot (point) in the graph. The position thereof is determined bythe relative values of FSC and SSC. Lymphocytes with a relatively smallsize and simple internal structure are displayed in the bottom leftsection, granulocytes with a large size and a granule inside aredisplayed in the top right section, and mononuclear cells with a largesize but a simple internal structure are displayed between thelymphocytes and the granulocytes, as populations separated from oneanother.

Fluorescent light refers to a light generated when a fluorescent pigmentlabeling a cell is excited by an irradiated laser beam and releasesenergy. A flow cytometer (e.g., product name: Becton & DickinsonFACSCalibur) typically irradiates a 488 nm single wavelength laser beamand a 635 nm single wavelength laser beam. A cell itself also has aproperty of emitting weak fluorescence (autofluorescence). However, whenactually attempting to specifically detect a molecule of a cell usingfluorescence, it is necessary to bind a fluorescent pigment to a cell orits molecule in some form in advance. For example, FITC (Fluoresceinisothiocyanate) absorbs a 488 nm excitation light and mainly emits a 530nm fluorescent light (green). Labeling of an antibody with FITC inadvance leads to a difference in the amount of antibodies bound inaccordance with the amount of antigens on the cell surface, resulting ina difference in the fluorescence intensity of FITC. Thus, the amount ofantigens that are present on the cell surface can be estimated.FACSCalibur that can be used as an example has four fluorescencedetectors installed, which can detect different fluorescence wavelengthregions. If multiple fluorescent pigments that emit light with differentwavelengths are prepared, up to four different antigens can besimultaneously detected. As a fluorescent pigment other than FITC whichis excited by a 488 nm single wavelength laser beam, PE (phycoerythrin)mainly emits a 585 nm fluorescent light, and PerCP (peridininchlorophyll protein) and PE-Cy5 (carbocyanin-5) mainly emit a 670 nmfluorescent light. APC (allophycocyanin), which is a fluorescent pigmentexcited by a 635 nm single wavelength laser beam, mainly emits a 670 nmfluorescent light. These fluorescent pigments are combined with variousantibodies and used in double or triple staining of cells. CD4, CD8,CD62L, CD25, Foxp3 molecules, and the like that are expressed on thesurface of T lymphocytes can be detected with a monoclonal antibody,which specifically react thereto.

Strictly speaking, there are two types of flow cytometers, i.e.,equipment which only analyzes cells and equipment capable of sortinganalyzed cells. The latter is called “FACS”. As used herein, “FACS” isan abbreviation of fluorescence-activated cell sorter and refers to anapparatus used in a method of analyzing a surface antigen of a free cellsuch as a lymphocyte using a laser beam or sorting a specific cell bythe presence/absence of a surface antigen or the like.

A result of flow cytometry can be displayed in a histogram, dot plot, orthe like.

As used herein, “histogram” refers to a graph representing intensity ofan optical signal of each parameter on the X axis and the cell count onthe Y axis in fluorescence measurement using a flow cytometer. 10thousand or more cells can be counted in total in such a form.

As used herein, “dot plot” refers to a plot of fluorescence intensity oftwo types of fluorescent pigments on the X and Y axes. In the double- ortriple-stained analysis, this can be analyzed using a display method inwhich the respective fluorescence intensity is placed on the X or Y axisand individual cells correspond to each point on a two dimensionalgraph.

For example, peripheral blood or bone marrow liquid is collected, andthen erythrocytes are removed by hemolytic method or specific gravitycentrifugation, then the residual is reacted with a fluorescentlylabeled antibody (antibody to antigen of interest and a control antibodythereof) and sufficiently washed for observation using flow cytometry.

The detected scattered light or fluorescence is converted to an electricsignal and analyzed by a computer. The result can distinguishlymphocytes, mononuclear cells, and granulocytes by representing theintensity of FSC as the cell size and the intensity of SSC asintracellular structure. The cell population of interest is gatedthereafter as needed to examine the antigen expressing manner in thecells.

In practicing the method of the present invention, those skilled in theart can suitably identify a surface marker of the shown cells tofractionate or count the cells.

CD antigens were agreed upon in an international workshop to beclassified (clustering) as clusters mainly by the biochemical feature(especially molecular weight) of an antigen recognized thereby. This iscalled CD classification. Many types of monoclonal antibodies thatrecognize a specific leukocyte differentiation antigen are named therebyunder a unified convention, which is CD followed by a number, i.e., CDnumber (i.e., CD1, CD2, and the like).

Typical examples of cell surface markers including CD markers that areused herein are explained hereinafter.

CD4 (6.2): binds to an MHC class II molecule on an antigen presentingcell and functions as a co-receptor of a T lymphocyte antigen receptorcomplex. CD4 is expressed in MHC class II restricted helper Tlymphocyte.

CD8 (6.4): is a dimeric protein with an S—S bond of α and β chains. CD8binds to an MHC class I molecule on an antigen presenting cell andfunctions as a co-receptor of a T lymphocyte antigen receptor complex.CD8 is expressed on MHC class I restricted killer T lymphocytes.

CD25: CD25 is a 55 kDa glycoprotein which is also known as a lowaffinity interleukin-2 receptor a chain (IL-2Rα). CD25 is also expressedin activated T-cells, B cells, and macrophages, and some non-activatedCD4⁺ T-cells, which act as regulatory T-cells. Thus, CD25 is utilized asa marker for regulatory T-cells.

CD62L: CD62L (L-selectin) is a molecule, which is required forrecognizing and homing a high endothelial venule (HEV) that is presentspecifically in lymphoid organs. Naive T-cells have this molecule toprepare for circulation through the lymphoid organs and antigenpresentation. Naive T-cells lose a homing molecule upon recognizing anantigen presented by dendritic cells in lymphoid organs is recognizedwith a T-cell receptor and being primed by effector T-cells. Thus,effector T-cells that have been primed by antigen recognition and clonedand expanded have the CD62L^(low) phenotype.

Foxp3: Foxp3 is a master transcription factor of regulatory T-cells(Treg), i.e., transcription factor playing an essential role in all ofdifferentiation/function expression/maintenance of differentiated statusof Tregs. Since expression is nearly specific to Treg, Foxp3 is commonlyused as a marker molecule for identifying Tregs. Foxp3 increases CD25 orCTLA4 expression, while suppressing the production of effector cytokines(IL-2, IFNγ, IL-4, IL-17, and the like).

PD-1 is deeply involved with the phenomenon of T cell exhaustion. Inshort, this phenomenon is attenuation of a T-cell reaction to antigensthat are present in large quantity and for an extended period of time.Even if naive T-cells become T-cells with a high level of effectorfunction due to priming by antigen presenting cells, the T-cells, uponexposure to large quantity antigen presentation for an extend period oftime, express immune checkpoint molecules PD-1→LAG-3→CD244, lose thefunction and ultimately result in apoptosis. Since cancer cells arepresent “in large quantity” and “for an extended period of time”, it canbe understood that this system is in effect.

(Effect of Preventing/Treating Cancer by CD62L^(low)CD4⁺ T-CellInfusion)

Another aspect of the present invention is a method of improving ormaintaining/sustaining a therapy effect of cancer immunotherapy byinfusion of a specific cell or a composition therefor.

CD62L^(low)CD4⁺ T-cells have been found to be critical in a response ofa subject to cancer immunotherapy. It is understood that the use of suchT-cells can improve or maintain responsiveness to cancer immunotherapyof a subject. One embodiment of the present invention is a compositioncomprising a CD62L^(low)CD4⁺ T-cell. A CD62L^(low)CD4⁺ T-cell or acomposition comprising the same is useful for concomitant use withcancer immunotherapy.

Although not wishing to be bound by any theory, a therapeutic effect dueto CD62L^(low)CD4⁺ T-cell infusion into a patient on whom a PD-1inhibitor and/or PD-L1 inhibitor does not achieve a sufficient effect ofpreventing/treating cancer can be understood as follows.

When PD-L1 expressed on the cancer cell surface binds to PD-1 expressedon the T-cell surface, an anti-tumor effect due to T-cells is suppressed(immune evasion mechanism by cancer cells). Anti-PD-1 antibodies areantibody molecules, which inhibit such a bond between PD-L1 and PD-1 andblock the immune evasion mechanism by cancer cells to allow exertion ofan anti-tumor effect by T-cells. Thus, it is understood that ananti-tumor effect due to inhibition of a PD-1/PD-L1 bond is primarilyexerted in the effector phase where T-cells attack tumor, while theeffect in the T-cell priming phase is low. In other words, it isdifficult for a PD-1 or PD-L1 inhibitor to exert an anti-tumor effectunless there are already T-cell priming and sufficient effector T-cells.The maximum anti-tumor effect is exerted by anti-PD-1 antibodies onabout 20% to 30% of cancer patients, but a T-cell immunity statusrequired for an anti-PD-1 antibody to exert an anti-tumor effect and amethod of evaluating such an immunity status were unknown.

CD62L (L-selectin) is a “homing receptor” of lymphocytes. CD62L isexpressed on the cell surface of naive T-cells and promotes themigration thereof into the lymph node. When naive T-cells in the lymphnode is subjected to antigen stimulation by an antigen presenting cell,the cells are activated into effector T-cells, while the CD62Lexpression level decreases (CD62L^(low)), and differentiate into CD4⁺T-cells (helper T-cells) or CD8⁺ T-cells (cytotoxic T-cells). Theinventors have discovered that CD62L^(low) T-cells are very effective asa method of identifying T-cells primed by cancer antigens undercircumstances where cancer antigens are unknown and cancer antigenspecific T-cells cannot be identified. In a mouse model, CD62L^(low)T-cells separated from a tumor regional lymph node can be adoptivelyinfused to heal a cancer bearing mouse. Use of effector T-cellsseparated in this manner achieved a greater anti-tumor effect byintroducing CD62L^(low)CD4⁺ T-cells (Example 4 and FIGS. 14A-B). Mostcancer cells including the tumor system used in this experiment do notexpress MHC class II antigens. Thus, it is understood that the highanti-tumor effect of CD62L^(low)CD4⁺ T-cells is attained not byinfluencing a direct cytocidal function, but instead a function ofantigen presenting cells such as dendritic cells to orchestrate theentire T-cell immunity. An excellent anti-tumor effect was also achievedwhen CD62L^(low)CD8⁺ T-cells were used together with CD62L^(low)CD4⁺T-cells (Example 4 and FIGS. 14A-B).

The inventors have discovered that the ratio of CD62L^(low)CD4⁺ T-cellsin all T-cells is clearly correlated with an anti-tumor effect ofanti-PD-1 antibodies, i.e., it is essential to comprise CD62L^(low)CD4⁺T-cells, which are T-cells exerting an anti-tumor effect, in order toexert an anti-tumor effect with anti-PD-1 antibodies.

Although not wishing to be bound by any theory, it is understood fromthis finding that T-cell immunity, which is ordinarily sufficient toexert an anti-tumor effect, is prepared, but immunity is evaded due toattenuation of antigen recognition signals by PD-1/PD-L1 in cancerpatients comprising many CD62L^(low)CD4⁺ T-cells. CD62L^(low)CD4⁺T-cells activate antigen presenting cells such as dendritic cells toactivate a priming phase. Further, primed CD8⁺ T-cells need to besubjected to antigen presentation from local antigen presenting cellsthat have been activated by effector CD4⁺ T-cells in order to acquire acytotoxic function. In this regard, it is understood that a PD-1/PD-L1binding inhibitor, which restores attenuation of an antigen recognitionsignal primarily in the effector phase is complementary to the functionof effector CD4⁺ T-cells. It is understood that the antigen presentingcell function, which should present cancer antigens, is still suppressedeven if the immune evasion mechanism is blocked with an anti-PD-1antibody in patients who do not comprise a large quantity ofCD62L^(low)CD4⁺ T-cells, resulting in an unsatisfactory anti-tumoreffect.

In view of the above, it is understood that administration ofCD62L^(low)CD4⁺ T-cells can exert an anti-tumor effect with an anti-PD-1antibody on patients with low CD62L^(low)CD4⁺ T-cell count resulting inan anti-tumor effect is not being exerted with an anti-PD-1 antibody.

(Manufacture and Use of Cell Containing Composition)

A method of manufacturing a composition comprising a CD62L^(low)CD4⁺T-cell can comprising purifying CD62L^(low)CD4⁺ T-cells from a T-cellpopulation derived from a human. The purifying may comprise removing aCD62L high expression cell from a T-cell population (negative selection)Purification of CD62L^(low)CD4⁺ T-cells by negative selection using anantibody and/or magnetic beads, or the like is preferable becauseimpurities such as an antibody or magnetic beads do not remain on a cellto be used.

One embodiment of the present invention is a kit comprising a substance,which specifically binds to CD62L, for purifying CD62L^(low)CD4⁺T-cells. Examples of substances which specifically bind to CD62Linclude, but are not limited to, antibodies that are specific to CD62L.Those skilled in the art can isolate and expand a specific T-cellsubpopulation disclosed herein in accordance with a method disclosedherein, e.g., flow cytometry. In one embodiment, the compositiondisclosed herein provides a CD4⁺CD62L^(low) T-cell.

T lymphocytes can be collected in accordance with a known technique andconcentrated or drained by a known technique such as flow cytometryand/or affinity binding to an antibody such as immunomagnetic selection.After the concentration and/or draining step, in vitro expansion ofdesired T lymphocytes can be performed in accordance with a knowntechnique (including, but not limited to, the technique disclosed inU.S. Pat. No. 6,040,177 by Riddell et al.) or a variation thereof thatwill be apparent to those skilled in the art.

For example, a desired T-cell population or subpopulation may beexpanded by adding a first T lymphocyte population to a medium in vitro,then adding a feeder cell to the medium (e.g., so that the produced cellpopulation contains at least about 5, 10, 20, 40 or more feeder cellsfor every T lymphocyte in the first population to be expanded), andincubating the culture (e.g., for a sufficient time to increase thenumber of T-cells). The culture can be typically incubated underconditions, such as temperature, that are suitable for the expansion ofT lymphocytes. For growth of human T lymphocytes, the temperature isgenerally, for example, at least about 25° C., preferably at least about30° C., and more preferably about 37° C.

Cells can be separated and/or expanded, and then stored as needed andadministered to a subject thereafter in accordance with the methoddisclosed herein or a method that is well known in the art.

The amount of cells of interested (e.g., CD62L^(low)CD4⁺ T-cell) in thecomposition comprising cell of the present invention, can beappropriately determined by the skilled in the art such that theintended effect is exerted, for example, may be at least 2×10⁸,preferably at least 6×10⁸, more preferably at least 2×10⁹, for humanadministration. A composition comprising cells disclosed herein cancomprise a pharmaceutically acceptable carrier or excipient in additionto a cell of interest (e.g., CD62L^(low)CD4⁺ T-cell). As used herein,“pharmaceutically acceptable” means approved by a government supervisoryauthority or listed in the Pharmacopoeia or other commonly recognizedpharmacopoeia for use in animals, or more specifically humans. As usedherein, “carrier” refers to a culture, infusion solution, perfusate,diluent, adjuvant, excipient, or vehicle, which is administered with atherapeutic agent. The composition comprising cells of the presentinvention comprises cells as the main ingredient, so that a carrier canpreferably maintain cells such as culture, infusion solution orperfusate. For example, when a pharmaceutical composition isintravenously administered, saline and aqueous dextrose are preferredcarriers. Preferably, an aqueous saline solution and aqueous dextroseand glycerol solution are used as a liquid carrier of an injectablesolution. When a medicament is orally administered, water is a preferredcarrier. Examples of suitable excipients include light anhydrous silicicacid, crystalline cellulose, mannitol, starch, glucose, lactose,sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodiumstearate, glyceryl monostearate, talc, sodium chloride, powdered skimmilk, glycerol, propylene, glycol, water, ethanol, carmellose calcium,carmellose sodium, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinyl acetal diethylamino acetate,polyvinylpyrrolidone, gelatin, middle chain fatty acid triglyceride,polyoxyethylene hydrogenated castor oil 60, refined sugar,carboxymethylcellulose, cornstarch, inorganic salt, and the like. Whendesired, a composition can also contain a small amount of humectant oremulsifier, or a pH buffering agent. These compositions can be in a formof a solution, suspension, emulsion, tablet, pill, capsule, powder,sustained release formulation or the like. A composition can also beformulated as a suppository by using a traditional binding agent and acarrier such as a triglyceride. An oral formulation can also comprise astandard carrier such as a medical grade mannitol, lactose, starch,magnesium stearate, saccharine sodium, cellulose or magnesium carbonate.Examples of suitable carriers are disclosed in E. W. Martin, Remington'sPharmaceutical Sciences (Mark Publishing Company, Easton, U.S.A). Such acomposition contains a therapeutically effective amount of a therapeuticagent, preferably in a refined form, together with a suitable amount ofcarrier to be provided in a form that is suitable for administration toa patient. A formulation must be suitable to the manner ofadministration. A formulation may additionally comprise, for example, asurfactant, excipient, coloring agent, flavoring agent, preservative,stabilizer, buffering agent, solubilizing agent, isotonizing agent,binding agent, disintegrant, lubricant, flow promoter, corrigent or thelike.

Preferred Embodiments

One embodiment of the present invention is a method of using an amountselected from:

an amount of a CD4⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;an amount of a dendritic cell subpopulation correlated with a dendriticcell stimulation by a CD4⁺ T-cell in an anti-tumor immune response;an amount of a CD8⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;an amount of regulatory T-cells or a CD4⁺ T-cell subpopulationcorrelated with regulatory T-cells; andan amount of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation;in a subject as a variable (indicator) of a formula for predicting aresponse to cancer immunotherapy of the subject. In one embodiment, avariable (indicator) of a formula for predicting a response to cancerimmunotherapy of a subject is selected from the group consisting of:an amount of a CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a CCR7⁻CD4⁺ T-cell subpopulation;an amount of a CD45RA⁻CD4⁺ T-cell subpopulation;an amount of a CD45RO⁺CD4⁺ T-cell subpopulation;an amount of a LAG-3⁺CD62L^(low)CD4⁺ T-cell subpopulation;an amount of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a CCR4⁺CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD127⁺CD25⁺CD4⁺ T-cell subpopulation; an amount of aCD45RA⁻ Foxp3+CD4⁺ T-cell subpopulation;an amount of a CD4⁺CD25⁺ T-cell subpopulation;an amount of a CD4⁺Foxp3⁺ T-cell subpopulation;an amount of a Foxp3⁺CD25⁺CD4⁺ T-cell subpopulation;an amount of an HLA-DR⁺ dendritic cell subpopulation;an amount of a CD80⁺ dendritic cell subpopulation;an amount of a CD86⁺ dendritic cell subpopulation;an amount of a PD-L1⁺ dendritic cell subpopulation;an amount of a CD62L^(low)CD8⁺ T-cell subpopulation;an amount of a CD137⁺CD8⁺ T-cell subpopulation; andan amount of a CD28⁺CD62L^(low)CD8⁺ T-cell subpopulation.

One embodiment of the present invention is a method of using a relativeamount selected from:

a relative amount of a CD4⁺ T-cell subpopulation correlated with adendritic cell stimulation in an anti-tumor immune response;a relative amount of a dendritic cell subpopulation correlated with adendritic cell stimulation by a CD4⁺ T-cell in an anti-tumor immuneresponse;a relative amount of a CD8⁺ T-cell subpopulation correlated with adendritic cell stimulation in an anti-tumor immune response;a relative amount of regulatory T-cells or a CD4⁺ T-cell subpopulationcorrelated with regulatory T-cells; anda relative amount of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation;in a subject as a variable (indicator) of a formula for predicting aresponse to cancer immunotherapy of the subject. In one embodiment, therelative amount, as a variable (indicator) of a formula for predicting aresponse to cancer immunotherapy of a subject, is selected from thegroup consisting of:a ratio of a CD62L^(low)CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CCR7⁻CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD45RA⁻CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD45RO⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a LAG-3⁺CD62L^(low) CD4⁺ T-cell subpopulation inCD62L^(low)CD4⁺ T-cells;a ratio of an ICOS⁺CD62L^(low) CD4⁺ T-cell subpopulation inCD62L^(low)CD4⁺ T-cells;a ratio of a CCR4⁺CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD127⁺CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD4⁺CD25⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD4⁺Foxp3⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a Foxp3⁺CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of an HLA-DR⁺ dendritic cell subpopulation in dendritic cells;a ratio of a CD80⁺ dendritic cell subpopulation in dendritic cells;a ratio of a CD86⁺ dendritic cell subpopulation in dendritic cells;a ratio of a PD-L1⁺ dendritic cell subpopulation in dendritic cells;a ratio of a CD62L^(low)CD8⁺ T-cell subpopulation in CD8⁺ T-cells;a ratio of a CD137⁺CD8⁺ T-cell subpopulation in CD8⁺ T-cells; andan ratio of a CD28⁺CD62L^(low)CD8⁺ T-cell subpopulation inCD62L^(low)CD8⁺ T-cells.

One embodiment of the present invention is a method of using an amountselected from:

an amount of a CD4⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;an amount of a dendritic cell subpopulation correlated with a dendriticcell stimulation by a CD4⁺ T-cell in an anti-tumor immune response;an amount of a CD8⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response; andan amount of regulatory T-cells or a CD4⁺ T-cell subpopulationcorrelated with regulatory T-cells;in a subject as a variable (indicator) of a formula for predicting aresponse to cancer immunotherapy of the subject, wherein an indicatorformula higher than a threshold value (ineffective group thresholdvalue) indicates that the subject is not a part of an ineffective groupto the cancer immunotherapy. In one embodiment, a variable (indicator)of a formula for predicting a response to cancer immunotherapy of asubject is selected from the group consisting of:an amount of a CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a LAG-3⁺CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a CCR7⁻CD4⁺ T-cell subpopulation;an amount of a CD45RA⁻CD4⁺ T-cell subpopulation;an amount of a CD45RO⁺CD4⁺ T-cell subpopulation;an amount of an HLA-DR⁺ dendritic cell subpopulation;an amount of a CD80⁺ dendritic cell subpopulation;an amount of a CD86⁺ dendritic cell subpopulation;an amount of a PD-L1⁺ dendritic cell subpopulation;an amount of a CD62L^(low)CD8⁺ T-cell subpopulation;an amount of a CD137⁺CD8⁺ T-cell subpopulation;an amount of a CD28⁺CD62L^(low)CD8⁺ T-cell subpopulation;an amount of a CD4⁺Foxp3⁺CD25⁺ T-cell subpopulation;an amount of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation;an amount of a CCR4⁺CD25⁺CD4⁺ T-cell subpopulation; andan amount of a CD127⁺CD25⁺CD4⁺ T-cell subpopulation;wherein an indicator formula higher than a threshold value (ineffectivegroup threshold value) indicates that the subject is not a part of anineffective group to the cancer immunotherapy.

One embodiment of the present invention is a method of using a relativeamount selected from:

a relative amount of a CD4⁺ T-cell subpopulation correlated with adendritic cell stimulation in an anti-tumor immune response;a relative amount of a dendritic cell subpopulation correlated with adendritic cell stimulation by a CD4⁺ T-cell in an anti-tumor immuneresponse;a relative amount of a CD8⁺ T-cell subpopulation correlated with adendritic cell stimulation in an anti-tumor immune response; anda relative amount of regulatory T-cells or a CD4⁺ T-cell subpopulationcorrelated with regulatory T-cells;in a subject as a variable (indicator) of a formula for predicting aresponse to cancer immunotherapy of the subject, wherein an indicatorformula higher than a threshold value (ineffective group thresholdvalue) indicates that the subject is not a part of an ineffective groupto the cancer immunotherapy. In one embodiment, the relative amount, asa variable (indicator) of a formula for predicting a response to cancerimmunotherapy of a subject is selected from the group consisting of:a ratio of a CD62L^(low)CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a LAG-3⁺CD62L^(low)CD4⁺ T-cell subpopulation inCD62L^(low)CD4⁺ T-cells;a ratio of a CCR7⁻CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD45RA⁻CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD45RO⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of an HLA-DR⁺ dendritic cell subpopulation in dendritic cells;a ratio of a CD80⁺ dendritic cell subpopulation in dendritic cells;a ratio of a CD86⁺ dendritic cell subpopulation in dendritic cells;a ratio of a PD-L1⁺ dendritic cell subpopulation in dendritic cells;a ratio of a CD62L^(low)CD8⁺ T-cell subpopulation in CD8⁺ T-cells;a ratio of a CD137⁺CD8⁺ T-cell subpopulation in CD8⁺ T-cells;an ratio of a CD28⁺CD62L^(low)CD8⁺ T-cell subpopulation inCD62L^(low)CD8⁺ T-cells;a ratio of a CD4⁺Foxp3⁺CD25⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CCR4⁺CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells; anda ratio of a CD127⁺CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;wherein an indicator formula higher than a threshold value (ineffectivegroup threshold value) indicates that the subject is not a part of anineffective group to the cancer immunotherapy.

One embodiment of the present invention is a method of predicting aresponse to cancer immunotherapy of a subject using amounts (X, Y)selected from:

an amount of a CD4⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response;an amount of a dendritic cell subpopulation correlated with a dendriticcell stimulation by a CD4⁺ T-cell in an anti-tumor immune response;an amount of a CD8⁺ T-cell subpopulation correlated with a dendriticcell stimulation in an anti-tumor immune response; andan amount of regulatory T-cells or a CD4⁺ T-cell subpopulationcorrelated with regulatory T-cells;as a variable (indicator) of formula F(X, Y), wherein formula F(X, Y)higher than a threshold value (ineffective group threshold value)indicates that the subject is not a part of an ineffective group to thecancer immunotherapy. In one embodiment, formula F(X, Y) can becalculated, with a variable (indicator) of a formula for predicting aresponse to cancer immunotherapy of a subject, which is a value selectedfrom the group consisting of:an amount of a CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a LAG-3⁺CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a CCR7⁻ CD4⁺ T-cell subpopulation;an amount of a CD45RA⁻CD4⁺ T-cell subpopulation;an amount of a CD45RO⁺CD4⁺ T-cell subpopulation;an amount of an HLA-DR⁺ dendritic cell subpopulation;an amount of a CD80⁺ dendritic cell subpopulation;an amount of a CD86⁺ dendritic cell subpopulation;an amount of a PD-L1⁺ dendritic cell subpopulation;an amount of a CD62L^(low)CD8⁺ T-cell subpopulation;an amount of a CD137⁺CD8⁺ T-cell subpopulation; andan amount of a CD28⁺CD62L^(low)CD8⁺ T-cell subpopulation as (X). In oneembodiment, F(X, Y) can be calculated, with a variable (indicator) of aformula for predicting a response to cancer immunotherapy of a subject,which is a value selected from the group consisting of:an amount of a CD4⁺Foxp3⁺CD25⁺ T-cell subpopulation;an amount of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation;an amount of a CCR4⁺CD25⁺CD4⁺ T-cell subpopulation; andan amount of a CD127⁺CD25⁺CD4⁺ T-cell subpopulation;as (Y). Formula F(X, Y) higher than a threshold value (ineffective groupthreshold value) indicates that the subject is not a part of anineffective group to the cancer immunotherapy.

One embodiment of the present invention is a method of predicting aresponse to cancer immunotherapy of a subject using amounts (X, Y)selected from:

a relative amount of a CD4⁺ T-cell subpopulation correlated with adendritic cell stimulation in an anti-tumor immune response;a relative amount of a dendritic cell subpopulation correlated with adendritic cell stimulation by a CD4⁺ T-cell in an anti-tumor immuneresponse;a relative amount of a CD8⁺ T-cell subpopulation correlated with adendritic cell stimulation in an anti-tumor immune response; anda relative amount of regulatory T-cells or a CD4⁺ T-cell subpopulationcorrelated with regulatory T-cells;in a subject as a variable (indicator) of formula F(X, Y), whereinformula F(X, Y) higher than a threshold value (ineffective groupthreshold value) indicates that the subject is not a part of anineffective group to the cancer immunotherapy. In one embodiment,formula F(X, Y) can be calculated, with a variable (indicator) of aformula for predicting a response to cancer immunotherapy of a subject,which is a value selected from the group consisting of:a ratio of a CD62L^(low)CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a LAG-3⁺CD62L^(low)CD4⁺ T-cell subpopulation inCD62L^(low)CD4⁺ T-cells;a ratio of a CCR7⁻CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD45RA⁻CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD45RO⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of an HLA-DR⁺ dendritic cell subpopulation in dendritic cells;a ratio of a CD80⁺ dendritic cell subpopulation in dendritic cells;a ratio of a CD86⁺ dendritic cell subpopulation in dendritic cells;a ratio of a PD-L1⁺ dendritic cell subpopulation in dendritic cells;a ratio of a CD62L^(low)CD8⁺ T-cell subpopulation in CD8⁺ T-cells;a ratio of a CD137⁺CD8⁺ T-cell subpopulation in CD8⁺ T-cells; andan ratio of a CD28⁺CD62L^(low)CD8⁺ T-cell subpopulation inCD62L^(low)CD8⁺ T-cells as (X). In one embodiment, formula F(X, Y) canbe calculated, with a variable (indicator) of a formula for predicting aresponse to cancer immunotherapy of a subject, which is a value selectedfrom the group consisting of:a ratio of a CD4⁺Foxp3⁺CD25⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CCR4⁺CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells; anda ratio of a CD127⁺CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells; as(Y). Formula F(X, Y) higher than a threshold value (ineffective groupthreshold value) indicates that the subject is not a part of anineffective group to the cancer immunotherapy.

Another embodiment of the present invention is a method of predicting aresponse to cancer immunotherapy of a subject, wherein formula F(X, Y)higher than a threshold value (ineffective group threshold value)indicates that the subject is not a part of an ineffective group to thecancer immunotherapy. Formula F(X, Y) can be calculated, with a variable(indicator) of a formula for predicting a response to cancerimmunotherapy of a subject, which is a value selected from the groupconsisting of:

an amount of a CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a LAG-3⁺CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a CCR7⁻CD4⁺ T-cell subpopulation;an amount of a CD45RA⁻CD4⁺ T-cell subpopulation;an amount of a CD80⁺ dendritic cell subpopulation;an amount of a CD62L^(low)CD8⁺ T-cell subpopulation;an amount of a CD137⁺CD8⁺ T-cell subpopulation; andan amount of a CD28⁺CD62L^(low)CD8⁺ T-cell subpopulationas (X), and a value selected form the group consisting of:an amount of a CD4⁺Foxp3⁺CD25⁺ T-cell subpopulation;an amount of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation; andan amount of a CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation;

as (Y).

Another embodiment of the present invention is a method of predicting aresponse to cancer immunotherapy of a subject, wherein formula F(X, Y)higher than a threshold value (ineffective group threshold value)indicates that the subject is not a part of an ineffective group to thecancer immunotherapy. Formula F(X, Y) can be calculated, with a variable(indicator) of a formula for predicting a response to cancerimmunotherapy of a subject, which is a value selected from the groupconsisting of:

a ratio of a CD62L^(low)CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a LAG-3⁺CD62L^(low)CD4⁺ T-cell subpopulation inCD62L^(low)CD4⁺ T-cells;a ratio of a CCR7⁻CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD45RA⁻CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD80⁺ dendritic cell subpopulation in dendritic cells;a ratio of a CD62L^(low)CD8⁺ T-cell subpopulation in CD8⁺ T-cells;a ratio of a CD137⁺CD8⁺ T-cell subpopulation in CD8⁺ T-cells; and anratio of a CD28⁺CD62L^(low)CD8⁺ T-cell subpopulation in CD62L^(low)CD8⁺T-cells as (X), anda ratio of a CD4⁺Foxp3⁺CD25⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;anda ratio of a CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells; as(Y).

Any function (F(X, Y)) of X and Y which monotonically increases withrespect to X and monotonically decreases with respect to Y can be usedas the aforementioned formula F(X, Y). Examples of formula F(X, Y)indicating responsiveness include F=X^(r)*Y^(s), wherein r and s are anyreal numbers. When X is positively correlated with responsiveness and Yis negatively correlated with responsiveness, it is preferable that r isa positive number and s is a negative number. Integers can be used for rand s for simplicity of the formula. For instance, F(X, Y) can berepresented as Xn*Ym, wherein n and m are any integers. Examination ofthe inventors has shown that responsiveness to cancer immunotherapy of asubject can be accurately predicted using a formula with r and s in therange of −3 to 3. Examples of preferred forms of the formula include,but are not limited to, X/Y, X²/Y, X*Y, and the like.

One embodiment of the present invention is a method of using an amountselected from:

an amount of regulatory T-cells or a CD4⁺ T-cell subpopulationcorrelated with regulatory T-cells;an amount of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation;an amount of an LAG-3⁺CD62L^(low)CD4⁺ T cell subpopulation; andan amount of an PD-1⁺CD62L^(low)CD4⁺ T cell subpopulation;in a subject determined to be not a part of an ineffective group as avariable (indicator) of a formula for predicting a response to cancerimmunotherapy of the subject, wherein an indicator formula higher than athreshold value (response group threshold value) indicates that thesubject is a part of a response group to the cancer immunotherapy. Inone embodiment, a variable (indicator) of a formula for predicting aresponse to cancer immunotherapy of a subject is selected from the groupconsisting of:an amount of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation;an amount of a CD4⁺CD25⁺ T-cell subpopulation;an amount of a CD4⁺Foxp3⁺ T-cell subpopulation;an amount of a CD4⁺Foxp3⁺CD25⁺ T-cell subpopulation;an amount of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation;an amount of a CCR4⁺CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD127⁺CD25⁺CD4⁺ T-cell subpopulation;an amount of an LAG-3⁺CD62L^(low)CD4⁺ T cell subpopulation; andan amount of an PD-1⁺CD62L^(low)CD4⁺ T cell subpopulation.

One embodiment of the present invention is a method of using a relativeamount selected from:

a relative amount of regulatory T-cells or a CD4⁺ T-cell subpopulationcorrelated with regulatory T-cells;a relative amount of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation;a relative amount of an LAG-3⁺CD62L^(low)CD4⁺ T cell subpopulation; anda relative amount of an PD-1⁺CD62L^(low)CD4⁺ T cell subpopulation;in a subject determined to be not a part of an ineffective group as avariable (indicator) of a formula for predicting a response to cancerimmunotherapy of the subject, wherein an indicator formula higher than athreshold value (response group threshold value) indicates that thesubject is a part of a response group to the cancer immunotherapy. Inone embodiment, a variable (indicator) of a formula for predicting aresponse to cancer immunotherapy of a subject is selected from the groupconsisting of:a ratio of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation inCD62L^(low)CD4⁺ T-cells;a ratio of a CD4⁺CD25⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD4⁺Foxp3⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD4⁺Foxp3⁺CD25⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CCR4⁺CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD127⁺CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;an ratio of an LAG-3⁺CD62L^(low)CD4⁺ T cell subpopulation in CD4⁺T-cells; andan ratio of an PD-1⁺CD62L^(low)CD4⁺ T cell subpopulation in CD4⁺T-cells.

One embodiment of the present invention is a method of using amounts (W,Z) selected from:

an amount of regulatory T-cells or a CD4⁺ T-cell subpopulationcorrelated with regulatory T-cells; andan amount of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation;in a subject determined to be not a part of an ineffective group asvariables (indicators) of formula J(W, Z) for predicting a response tocancer immunotherapy of the subject, wherein formula J(W, Z) higher thana threshold value (response group threshold value) indicates that thesubject is a part of a response group to the cancer immunotherapy. Inone embodiment, formula J(W, Z) can be calculated, with a variable(indicator) of a formula for predicting a response to cancerimmunotherapy of a subject, which isan amount of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulationas (Z) and a value selected from the group consisting of:an amount of a CD4⁺CD25⁺ T-cell subpopulation;an amount of a CD4⁺Foxp3⁺ T-cell subpopulation;an amount of a CD4⁺Foxp3⁺CD25⁺ T-cell subpopulation;an amount of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation;an amount of a CCR4⁺CD25⁺CD4⁺ T-cell subpopulation; andan amount of a CD127⁺CD25⁺CD4⁺ T-cell subpopulation;as (W). Formula J(W, Z) higher than a threshold value (response groupthreshold value) indicates that the subject is a part of a responsegroup to the cancer immunotherapy.

One embodiment of the present invention is a method of using amounts (Z,W) selected from:

a relative amount of regulatory T-cells or a CD4⁺ T-cell subpopulationcorrelated with regulatory T-cells; anda relative amount of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation;in a subject determined to be not a part of an ineffective group, asvariables (indicators) of formula J(W, Z) for predicting a response tocancer immunotherapy of the subject, wherein formula J(W, Z) higher thana threshold value (response group threshold value) indicates that thesubject is a part of a response group to the cancer immunotherapy. Inone embodiment, formula J(W, Z) can be calculated, with a variable(indicator) of a formula for predicting a response to cancerimmunotherapy of a subject, which isa ratio of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation inCD62L^(low)CD4⁺ T-cells as (Z) and a value selected from the groupconsisting of:a ratio of CD4⁺CD25⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD4⁺Foxp3⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD4⁺Foxp3⁺CD25⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CCR4⁺CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells; anda ratio of a CD127⁺CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;as (W). Formula J(W, Z) higher than a threshold value (response groupthreshold value) indicates that the subject is a part of a responsegroup to the cancer immunotherapy.

Another embodiment of the present invention is a method of predicting aresponse to cancer immunotherapy of a subject who has been determined tobe not a part of an ineffective group, wherein formula J(W, Z) higherthan a threshold value (response group threshold value) indicates thatthe subject is a part of a response group to the cancer immunotherapy.Formula J(W, Z) can be calculated, with variables (indicators) of aformula for predicting a response to cancer immunotherapy of a subject,which are:

an amount of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulationas (Z) and a value selected from the group consisting of:an amount of a CD4⁺CD25⁺ T-cell subpopulation;an amount of a CD4⁺Foxp3⁺ T-cell subpopulation;an amount of a CD4⁺Foxp3⁺CD25⁺ T-cell subpopulation;an amount of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation; andan amount of a CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation;

as (W).

Another embodiment of the present invention is a method of predicting aresponse to cancer immunotherapy of a subject who has been determined tobe not a part of an ineffective group, wherein formula J(W, Z) higherthan a threshold value (response group threshold value) indicates thatthe subject is a part of a response group to the cancer immunotherapy.Formula J(W, Z) can be calculated, with variables (indicators) of aformula for predicting a response to cancer immunotherapy of thesubject, which are:

a ratio of an ICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation inCD62L^(low)CD4⁺ T-cells;as (Z) and a value selected from the group consisting of:a ratio of a CD4⁺CD25⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD4⁺Foxp3⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD4⁺Foxp3⁺CD25⁺ T-cell subpopulation in CD4⁺ T-cells;a ratio of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;anda ratio of a CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation in CD4⁺ T-cells;

as (W).

Any function (J(Z, W)) of Z and W which monotonically increases withrespect to Z and monotonically increases with respect to W can be usedas the aforementioned formula J(Z, W). Examples of formula J(Z, W)indicating responsiveness include J=Z^(r)*W^(s), wherein r and s are anyreal numbers. When Z is positively correlated with responsiveness and Wis negatively correlated with responsiveness, it is preferable that r isa positive number and s is a negative number. Integers can be used for rand s for simplicity of the formula. For instance, J(Z, W) can berepresented as Z^(n)*W^(m), wherein n and m are any integers.Examination of the inventors have shown that responsiveness to cancerimmunotherapy of a subject can be accurately predicted using a formulawith r and s in the range of −5 to 6. Examples of preferred forms of theformula include, but are not limited to, Z/W, Z²/W, Z*W, Z*W⁵ and thelike.

The method disclosed herein, including those disclosed above, can beused to apply cancer immunotherapy to a subject who has been indicatedas not a part of an ineffective group with respect to the cancerimmunotherapy and/or as a part of a responsive group. Any cancerimmunotherapy disclosed herein can be used.

One embodiment provides a composition for treating cancer in a subjectwho has been indicated as not a part of an ineffective group withrespect to the cancer immunotherapy and/or as a part of a responsivegroup by using the method disclosed herein, including those disclosedabove. The composition can comprise any active ingredient disclosedherein and have any constitution disclosed herein.

(General Techniques)

Molecular biological approaches, biochemical approaches, andmicrobiological approaches used herein are well known and conventionalapproaches in the art that are disclosed in, for example, Sambrook J. etal. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harborand its 3rd Ed. (2001); Ausubel, F. M. (1987). Current Protocols inMolecular Biology, Greene Pub. Associates and Wiley-Interscience;Ausubel, F. M. (1989). Short Protocols in Molecular Biology: ACompendium of Methods from Current Protocols in Molecular Biology,Greene Pub. Associates and Wiley-Interscience; Innis, M. A. (1990). PCRProtocols: A Guide to Methods and Applications, Academic Press; Ausubel,F. M. (1992). Short Protocols in Molecular Biology: A Compendium ofMethods from Current Protocols in Molecular Biology, Greene Pub.Associates; Ausubel, F. M. (1995). Short Protocols in Molecular Biology:A Compendium of Methods from Current Protocols in Molecular Biology,Greene Pub. Associates; Innis, M. A. et al. (1995). PCR Strategies,Academic Press; Ausubel, F. M. (1999). Short Protocols in MolecularBiology: A Compendium of Methods from Current Protocols in MolecularBiology, Wiley, and annual updates; Sninsky, J. J. et al. (1999). PCRApplications: Protocols for Functional Genomics, Academic Press;Bessatsu Jikken Igaku [Experimental Medicine, Supplemental Volume],Idenshi Donyu Oyobi Hatsugen Kaiseki Jikken Ho [Experimental Methods forTransgenesis & Expression Analysis], Yodosha, 1997, and the like. Therelevant portions (which can be the entire document) of the abovedocuments are incorporated herein by reference.

As used herein, “or” is used when “at least one or more” of the listedmatters in the sentence can be employed. When explicitly describedherein as “within the range” of “two values”, the range also includesthe two values themselves.

Reference literatures such as scientific literatures, patents, andpatent applications cited herein are incorporated herein by reference tothe same extent that the entirety of each document is specificallydescribed.

As disclosed above, the present invention has been disclosed whileshowing preferred embodiments to facilitate understanding. The presentinvention is disclosed hereinafter based on Examples. The aforementioneddescription and the following Examples are not provided to limit thepresent invention, but for the sole purpose of exemplification. Thus,the scope of the present invention is not limited to the embodiments andExamples specifically described herein and is limited only by the scopeof claims.

EXAMPLES

The present invention is exemplified by the following Examples herein.

(1) [Example Demonstrating Enablement of the Invention of Markers]

Example 1: Therapeutic effect of anti-PD-1 antibody and T-cellpopulation composition This Example demonstrates that analysis ofCD62L^(low)CD4⁺ T-cells and Foxp3+CD25+CD4+ T-cells using peripheralblood can predict a therapeutic effect of therapy with an anti-PD-1antibody.

(2) [Example Demonstrating Enablement of Invention of Cell Infusion]

Example 2: Hypothetical example of infusing CD62L^(low) cells

Example 3: Follow up observation

The Example demonstrates that an anti-tumor immune response of a patientis correlated with the T-cell composition, i.e., increase in CD62L^(low)cells enhances the anti-tumor immune response in a patient undergoinganti-PD-1 therapy to reduce tumor.

Example 4: Cell infusion into mice

The Example demonstrates that the percentage of CD62L^(low) CD4⁺ T-cellincreases by infusing CD62L^(low)CD4⁺ T-cells in mice. An increase inthe percentage of CD62L^(low)CD4⁺ T-cell and Treg also enhancesanti-tumor immune responses in mice.

Example 5: Isolation/Expansion of CD62L^(low) cells

The Example demonstrates that CD62L^(low) cells can be successfullyisolated and expanded.

Example 1: Therapeutic Effect of Anti-PD-1 Antibody and T-CellPopulation Composition

1-1. Objective

The objective of this Example is to demonstrate that analysis ofCD62L^(low)CD4⁺ T-cells and Foxp3⁺CD25⁺CD4⁺ T-cells using peripheralblood can predict a therapeutic effect of therapy with an anti-PD-1antibody. The relationship between a therapeutic effect of an anti-PD-1antibody and T-cell population composition was investigated.

1-2. Materials and Methods

The effect of nivolumab therapy in non-small cell lung cancer patientswas studied in accordance with the protocol shown in FIG. 2.

Peripheral blood was collected the day before nivolumab therapy fromnon-small cell lung cancer patients who have already undergone therapy.

CT was administered for determining the effect at week 8 from the startof nivolumab therapy. Partial response (PR), stable (SD), andprogressive (PD) at this point were determined. The criteria ofdetermination was in accordance with RECIST ver. 1.1. The followingTable 1 shows the Characteristics of patients.

TABLE 1 Characteristic of patients (n = 44) Age  Median value 67  Range51-84 Sex-number (%)  Male 30 (68)  Female 14 (32) Histologicaldiagnosis-number (%)  Squamous 12 (27)  Non-squamous 32 (73) History ofSmoking-number (%)  Current or previous smoker 33 (75)  No history ofsmoking 11 (25) Pathological phase-number (%)  c-stage III  9 (20) c-stage IV 26 (59)  Post-op recurrence  9 (20) EGFR status-number (%) Wild-type 37 (84)  Mutant (exon 19del or L858R) 17 (16)

The composition of a peripheral blood T-cell population of subjects wasanalyzed as follows.

(1) Blood Collection

8 ml of blood was collected in a blood collecting tube for mononuclearcell separation (product name: BD Vacutainer® CPT™, BD Japan), which wasgently inverted and mixed at room temperature.

(2) Centrifugation (Separation of Mononuclear Cells by Specific GravityCentrifugation)

After blood collection, BD Vacutainer® CPT™ was centrifuged at 1500 to1800×g for 15 minutes (centrifuge name/manufacturer: Kubota).

(3) Collection

About half of the plasma layer was aspirated so as not to disturb thecell layer above a gel barrier. The cell layer above the gel barrier wascollected with a Pasteur pipette and transferred into a 50 ml tube(Falcon tube or the like). A phosphate-buffered balanced salt solution(10% FBS PBS) supplemented with 10% fetal bovine serum was added so thatthe mixture was 30 ml or greater. The mixture was centrifuged (4° C.,400 to 450 g x 5 minutes) and washed twice.

(4) Cell Count

After completion of the first washing/centrifugation, 10 ml of PBSsupplemented with 10% FBS (inactivated at 56° C. in 30 minutes) wasadded to resuspend cells. 50 μl of cell suspension in a centrifuge tubewas collected. 0.1% trypan blue solution (50 μl) and the cell suspensionwere stirred. The cells were placed in an Improved Neubauerhemocytometer to count the cells.

(5) Freezing

After completion of the second washing/centrifugation, CELLBANKER™ 2(Takara Bio) was used. Cells were resuspended in 5×10⁵ to 5×10⁶/ml, andtransferred into a 2.0 ml cryogenic vial (Corning). After treatment, thecells were promptly frozen in a −80° C. deep freezer (Panasonic). After24 hours and within one week of the above treatment, the cells weretransferred into liquid nitrogen (under liquid phase).

(6) Culture

The frozen cells were adjusted to be 1 to 5×10⁵/ml in an RPMI 1640medium (FBS 10%) and cultured for 24 to 36 hours in a T-25 cell cultureflask at 37° C. with 5% CO′.

(7) Cell Adjustment

Cell culture was collected in a 15 ml centrifuge tube and wascentrifuged at 1500 rpm for 10 minutes to gather the cells at the bottomof the centrifuge tube. After the centrifugation, the supernatant wasremoved. 10 ml of FACS buffer was added to cell pellets to resuspend thecells with a pipette. The cells were against centrifuged at 1500 rpm for10 minutes, and then the supernatant was aspirated. The cells werecounted and adjusted so that the final cell concentration was 1.0×10⁶cells/ml. FACS buffer: 2% FBS, 0.05% Azide in PBS.

(8) Antibody Reaction

Suspension of peripheral blood mononuclear cells was placed in each FACStube at 0.5 ml (5×10⁵ cells would be in each tube). The tube wascentrifuged with a centrifuge at 1500 rpm for 5 minutes. The cellpellets were left while aspirating and removing only the supernatant.

Tube 1

20 μl of FITC labeled anti-human CD4 antibody (25 μg/ml)20 μl of PE labeled anti-human CD62L antibody (5 μg/ml)20 μl of PE-Cy5 labeled anti-human CD8 antibody (5 μg/ml)

An antibody solution and cell suspension were stirred and mixed. Thetubes were maintained at 4° C. After 30 minutes, 1 ml of FACS buffer wasadded to each tube with a Komagome pipette, and the mixture wascentrifuged with a centrifuge at 1500 rpm for 5 minutes. The supernatantwas aspirated and removed. 0.5 ml of 1% paraformaldehyde was added toeach tube, from which the supernatant was aspirated while leaving onlythe cell pellets, to suspend the cells.

Tube 2

20 μl of FITC labeled anti-human CD4 antibody (25 μg/ml)20 μl of PE-Cy5 labeled anti-human CD25 antibody (5 μg/ml)

An antibody solution and cell suspension were stirred and mixed. Thetubes were maintained at 4° C. After 30 minutes, 1 ml of FACS buffer wasadded to each tube, and the mixture was centrifuged with a centrifuge at1500 rpm for 5 minutes. The supernatant was aspirated and removed.Intracellular Fixation and Permeabilization buffer Set™ (eBioscience)was used for cytoplasmic staining with 3 μl of PE labeled anti-humanFOXP3 antibodies (500 μg/ml). 0.5 ml of 1% paraformaldehyde was added toeach tube, from which the supernatant was aspirated while leaving onlythe cell pellets, to suspend the cells.

(9) Analysis by Flow Cytometry (Product Name: FACS Calibur™; BD Japan)

Measurement of Samples

Fluorescence of tubes 1 and 2 are measured.

Incorporation of analysis data for 30,000 cells

Analysis

STEP 1 Tube 1 is analyzed to identify a lymphocyte region usingtwo-dimensional analysis using FSC or SSC. Cells gated in the lymphocyteregion are further gated with respect to CD4⁺fraction to obtain ahistogram plot of CD62L (cell count in the blue region)

STEP 2 Tube 2 is analyzed to obtain two-dimensional analysis data withFoxp3 and CD25, which are gated in the lymphocyte region and CD4⁺region.

(Cell Count in the Orange Region)

STEP 3 Calculation of the ratio of CD62L^(low)CD4⁺/Foxp3⁺CD25⁺CD4⁺

Formula Cell count in STEP 1/cell count in STEP 2

FIG. 1 shows an example of a result for cell fractions in flowcytometry. It should be noted that mRNA was measured with a microarraybetween CD62L^(low) and CD62L^(high). While the present Examplefractionates cells using flow cytometry, other separation methods canalso be used.

(Determination)

If lower than a predetermined value, Progressive Disease is predicted,for which drug is not effective.

If higher than a predetermined value, go to STEP 4

STEP 4

Formula Cell count in the orange region/cell count for R1 and R2 in STEP1×100(%)

(Determination)

If lower than a predetermined value, Stable Disease (SD) is predicted.

If higher than a predetermined value, Partial Response (PR) ispredicted.

Statistical analysis was conducted on the relationship between theresulting T-cell population composition and observed therapeutic effect.

1-3. Results

The following Table 2 shows the observed therapeutic effect on patients.

TABLE 2 Response to nivolumab Objective response in 8 weeks-number (%)Complete or partial response 11 (25) Stable 19 (43) Progressive 14 (32)

Confirmed complete and partial responses were evaluated in accordancewith the Response Evaluation Criteria in Solid Tumors, version 1.1 bythe testers.

The ratio of therapeutic effect observed in this Example isapproximately the same as the response rate obtained in the phase IIIclinical trial called checkmate 017. Thus, it is understood that thereis no bias in a response to nivolumab.

As shown in FIGS. 3A-3D, there is no significant difference in theperipheral blood white blood cell count, lymphocyte count, CD4⁺cellpercentage, or CD8⁺cell percentage between PR+SD groups and PD groups.The subject population in this Example did not comprise a completeresponse (CR) group. If a CR group were present, the CR group would beidentified as a part of the PR group of the present invention.

The result shows that the percentage of CD62^(low) cells in CD8 cellswas significantly lower in PD groups (FIG. 4A). However, the percentageof PR+SD group and PD group overlaps over a wide range, where P=0.0138in a significance test. In contrast, the percentage of CD62^(low) cellsin CD4⁺cells was completely different between the PR+SD group and the PDgroup with almost no overlap (FIG. 4B). Meanwhile, the result shows thatthe percentage of CD25⁺Foxp3⁺cells, which are regulatory T-cells in CD4+cells was significantly higher in the PD group (FIG. 4C). It is theconsensus in the art that the CD25⁺Foxp3⁺CD4⁺cell fraction can beconsidered a regulatory T-cell fraction.

Furthermore, results of analyzing the correlation among three T-cellsubpopulations with a difference between PD group and the PR+SD groupare shown in panels D and E of FIG. 4. A strong correlation was foundbetween the percentage of CD62L^(low)CD8⁺and the percentage ofCD62L^(low)CD4⁺(FIG. 4D). As biological significance, the CD8⁺effectorcount is suggested to be regulated by CD4⁺effectors. This shows that itis preferable to use only one of them as a biomarker. It is demonstratedthat use of the percentage of CD62L^(low)CD4⁺, which has a very small pvalue, as a biomarker for the effector side is very useful in theprediction of a therapeutic effect of an immune checkpoint inhibitor.

Furthermore, a correlation was not found between regulatory T-cells andthe percentage of CD62L^(low)CD4⁺. This indicates that the respectivecell counts are regulated by different mechanisms. It is understood thatthe precision of predicting a therapeutic effect can be enhanced byusing both in combination as a biomarker.

FIGS. 5 to 12 show the results of further examining a parameter whichcan be used as a biomarker.

A very good result of 92.6% sensitivity and 96.7% specificity wasobtained with 19.4% as the threshold value, even by using only thepercentage of CD62L^(low)CD4+ with a large difference (FIG. 5). Thesensitivity and specificity for various threshold values are shown inFIG. 6.

The precision of prediction using a relative value of regulator T-cellsand the percentage of CD62L^(low)CD4+ was examined. FIG. 7 shows theresults of a ratio (X/Y) of two factors that move differently in a PDgroup as a numerator and a denominator when using the percentage ofCD62L^(low) CD4+ as X and the percentage of CD25+Foxp3+CD4+ as Y. It isunderstood that use of this indicator can clearly distinguish a patientin which regulatory T-cells have significantly increased so that ananti-tumor effect is no longer observed. FIG. 8 shows sensitivity andspecificity for various threshold values. It is understood that a markerwith specificity of 100% and sensitivity of 71.4% is obtained when athreshold value is 7.35.

For a formula using a combination of these factors, a suitable formulawas examined using logistic regression from results in a sample of N=40while considering the weighting for these factors with respect to theeffect on therapeutic effects. A logistic regression model was used tofind a coefficient, resulting in deriving the formula of X^(2.475)/Y(FIG. 27). It is understood that responsiveness can be accuratelypredicted by using a formula with a coefficient in the vicinity thereof(X²⁻³/Y). For example, it is understood that formulas such as X²/Y andX³/Y can be used.

FIGS. 9 and 10 show results of squaring the percentage ofCD62L^(low)CD4+ and using X²/Y as the relative value of X and Y inparticular. It is understood that this can be utilized as a very goodbiomarker with sensitivity and specificity of 100%. FIG. 10 showssensitivity and specificity for various threshold values. It isunderstood that this can be utilized as a very good biomarker withsensitivity and specificity of 100% when using this value with athreshold value of 174.3.

FIG. 11 shows a result of examining a biomarker that can predict PR andSD after determining a PD group. It was unexpectedly discovered thatthere is a difference in not the percentage of CD62L^(low)CD4+, but inthe percentage of CD25⁺Foxp3⁺CD4⁺cells. Since CD25⁺Foxp3⁺CD4⁺cells areTregs having immunosuppressive action, the percentage ofCD25⁺Foxp3⁺CD4⁺being higher in a PR group with a greater anti-tumorimmune response was an unexpected result.

PR and SD were able to be identified with sensitivity of 52.8% andspecificity of 100% with a threshold value of the percentage ofCD25⁺Foxp3⁺CD4⁺cells at 2.05% (FIGS. 11A-11B). FIG. 12 shows sensitivityand specificity for various threshold values.

Although not wishing to be bound by any theory, the mechanism ofpredicting a clinical effect of cancer immunotherapy in the presentinvention can be understood as follows. *It is understood that CD4⁺T-cells transmit an instruction to dendritic cells via an MEW class Imolecule, and the dendritic cells receiving the instruction stimulateCD8⁺ T-cells via an MEW class II molecule. These CD4⁺ T-cells encompasseffector T-cells (e.g., CD62L^(low)CD4⁺ T-cells) and regulatory T-cells(e.g., Foxp3⁺CD25⁺ T-cells). Meanwhile, the present invention predicts aclinical effect of cancer immunotherapy by evaluating the balance ofboth CD62L^(low) CD4⁺ T-cells and Foxp3⁺CD25⁺ T-cells. CD62L(L-selectin) is a molecule required for recognizing and homing a highendothelial venule (HEV) that is present specifically in lymphoidorgans. Since naive T-cells, when stimulated by antigen presentingcells, are primed by effector T-cells so that CD62L expressiondecreases, homing is no longer performed by effector T-cells. Examplesof markers of effector T-cell priming of naive T-cells include CCR7 asin CD62L. As a result of priming, the expression level of CCR7decreases. Thus, CCR7 can be used instead of CD62L^(low). For example,CCR7¹⁰R′CD4⁺ T-cells and/or CCR7⁻CD4⁺ T-cells can be used instead of (orin addition to) CD62L^(low)CD4+ T-cells. Examples of cell subpopulationsthat can be used as an indicator of effector T-cells include, but arenot limited to, subpopulations selected from the group consisting ofCD62L^(low)CD4⁺ T-cell subpopulation, CCR7⁻CD4⁺ T-cell subpopulation,LAG-3⁺CD62L^(low) CD4⁺ T-cell subpopulation, ICOS⁺CD62L^(low) CD4⁺T-cell subpopulation, CD45RA⁻CD4⁺ T-cell subpopulation, CD45RO⁺CD4⁺T-cell subpopulation, HLA-DR⁺ dendritic cell subpopulation, CD80⁺dendritic cell subpopulation, CD86⁺ dendritic cell subpopulation, PD-L1⁺dendritic cell subpopulation, CD62L^(low)CD8⁺ T-cell subpopulation, andCD137⁺CD8⁺ T-cell subpopulation. The amount (absolute amount) and/orratio (relative amount) of these cell subpopulations can be utilized asan indicator of effector T-cells.

Examples of cell subpopulations that can be used as an indicator ofregulatory T-cells include, but are not limited to, cell subpopulationsselected from the group consisting of: an amount of a CCR4⁺CD25⁺CD4⁺T-cell subpopulation; an amount of a CD62L^(high)CD25⁺CD4⁺ T-cellsubpopulation; an amount of a CD127⁺CD25⁺CD4⁺ T-cell subpopulation; anamount of a CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation; an amount of aCD4⁺Foxp3⁺CD25⁺ T-cell subpopulation; and an amount of anICOS⁺CD62L^(low)CD4⁺ T-cell subpopulation. The amount (absolute amount)and/or ratio (relative amount) of these cell subpopulations can beutilized as an indicator of regulatory T-cells.

Example 2: Cell Therapy for Improving or Maintaining and Sustaining aTherapeutic Effect of Cancer Immunotherapy

Before starting therapy with cancer immunotherapy, CD62L^(low)CD4⁺T-cells are isolated from a peripheral blood sample of a subject andstored. The isolated CD62L^(low)CD4⁺ T-cells are expanded ex vivo (“exvivo expansion” in FIG. 13). Isolated CD62L^(low) CD4⁺ T-cells can befrozen and stored.

When a subject is determined as not a part of an ineffective group bythe procedure shown in Example 1 or the like, cancer immunotherapy suchas therapy using an anti-PD-1 antibody such as nivolumab is applied whenthe ratio of CD62L^(low)CD4⁺ T-cells/CD25⁺Foxp3⁺CD4⁺ T-cells is high, asshown in the top drawing of FIG. 13. During therapy, the CD4⁺ T-cellcomposition of a subject is monitored by the approach disclosed inExample 1.

In this regard, upon a decrease in an indicator such asCD62L^(low)CD4⁺percentage/CD25⁺Foxp3⁺CD4⁺cell percentage in the CD4⁺T-cell composition of a subject to reach an immunological condition ofan ineffective group, CD62L^(low)CD4⁺ T-cells expanded ex vivo can beinfused to recover the original immunological condition to sustain theeffect of cancer immunotherapy.

Storage/culturing cost can be minimized by culturing and infusing onlyCD62L^(low)CD4⁺ T-cells. This is more economical than continuing only animmune checkpoint inhibitor such as an anti-PD-1 antibody every twoweeks.

A subject who has a low indicator such as CD62L^(low)CD4+percentage/CD25⁺Foxp3⁺CD4⁺cell percentage in the CD4⁺ T-cell compositionof the subject and is determined as a part of an ineffective group (forexample, low ratio of CD62L^(low)CD4⁺ T-cells/CD25⁺Foxp3⁺CD4⁺ T-cells asin the bottom drawing of FIG. 13) is infused with CD62L^(low)CD4⁺T-cells, which have been isolated from the subject and expanded ex vivo,to change the immunological condition to response group, and then cancerimmunotherapy with an anti-PD-1 antibody such as nivolumab isadministered. An anti-tumor immune response of cancer immunotherapy canbe induced thereby even in subjects who have not been able to benefitfrom cancer immunotherapy with an anti-PD-1 antibody.

Example 3: Follow-Up Observation

Seven patients were subjected to follow-up to observe the percentage ofCD62L^(low)CD4⁺ T-cells. Peripheral blood mononuclear cells wereanalyzed every four weeks.

The results are shown in the following Table 3. Each of 1 to 7represents a result for different patients.

TABLE 3 % CD4 T cells CD62Llow CD4 1 29.6 23.6 32.5 14.9 2 60.5 25.854.4 39.9 3 40.4 43.7 43.3 39.8 44.8 39.7 41.2 34.4 4 30.3 25 33.3 28.431.9 24.3 26.4 28.5 5 39 30.2 34.5 39.3 6 36 24.6 33.8 32.6 7 34.3 24.624.7 30.2

Tumor regression was observed in patient 1 in the early stages ofstarting nivolumab therapy, but there was swelling in the cervical lymphnode for a period of time. While PD was suspected, the swelling wasreduced as of the evaluation CT after 8 weeks so that the patient wasdetermined as PR. When an increase in tumor size was observed, thepercentage of CD62L^(low)CD4⁺ T-cells decreased. When the tumor againregressed, the percentage of CD62L^(low)CD4⁺ T-cells was again elevated.All other subjects maintained a high percentage of CD62L^(low)CD4⁺T-cells from before the therapy, so that they were determined as PR orSD.

Combined with the findings in Example 1, it is understood that a subjectwould be unresponsive when the percentage of CD62L^(low)CD4⁺is less than19.4%, but it is understood that a subject would be responsive when thepercentage of CD62L^(low)CD4⁺ T-cells recovers again.

Example 4: Cell Infusion to Mice

Tumor model mice were infused with cells having the composition of 2×10⁶CD62L^(low)CD4⁺/5×10⁶ CD62L^(low)CD8⁺(FIG. 14A “●”), 5×10⁶CD62L^(low)CD8⁺(FIG. 14A “Δ”), and 1×10⁶ CD62L^(low)CD4⁺(FIG. 14B “●”).The development in tumor size over time was then observed.

The ratio of (CD62L^(low) cells in CD4⁺ T-cells)/(CD62L^(high)CD25⁺cellsin CD4⁺ T-cells) in the spleen on day 13 after tumor seeding wasmeasured in a group infused with cells, i.e.,2×10⁶CD62L^(low)CD4⁺/5×10⁶CD62L^(low)CD8⁺(FIG. 14A “●”) or 5×10⁶CD62L^(low)CD8⁺(FIG. 14A “Δ”), and a group without cell infusion (FIG.14A “∘”). In a group infused with 1×10⁶ CD62L^(low)CD4⁺cells (FIG. 14B“●”), the ratio of (CD62L^(low) cells in CD4⁺T-cells)/(CD62L^(high)CD25⁺cells in CD4⁺ T-cells) in the spleen wasmeasured over time. T-cell analysis of peripheral blood is challengingin mice. As an alternative, a common spleen cell analysis is used.T-cell analysis in mouse spleen is considered equivalent to PBMC inhumans. The T-cell fraction of CD4⁺CD62L^(high)CD25⁺is a fractioncomprising regulatory T-cells (Treg).

FIG. 14 shows the results. In the analysis of the T-cell composition onday 13, the ratio of (CD62L^(low) cells in CD4⁺T-cells)/(CD62L^(high)CD25⁺cells in CD4⁺ T-cells) is 10.6 in a groupinfused with 2×10⁶CD62L^(low)CD4+/5×10⁶CD62L^(low)CD8+ cells, 2.94 in agroup infused with 5×10⁶ CD62L^(low)CD8⁺cells, and 2.70 in a groupwithout infusion of cells. It is understood that infusion ofCD62L^(low)CD4+ cells increases the percentage of CD62L^(low)CD4⁺in theT-cell composition. Furthermore, a significant tumor regression isobserved in a group infused with 2×10⁶CD62L^(low)CD4+/5×10⁶CD62L^(low)CD8+ cells with a high ratio of (CD62L^(low) cells in CD4⁺T-cells)/(CD62L^(high)CD25⁺cells in CD4⁺ T-cells) (FIG. 14A “●”).

The above results show that an anti-tumor effect is achieved by infusingCD62L^(low)CD4⁺cells and by infusing a mixture of CD62L^(low)CD4⁺cellsand CD62L^(low)CD8⁺cells.

It can be understood that in a group infused with 1×10⁶CD62L^(low)CD4⁺cells, the ratio of CD62L^(low) cells in CD4⁺T-cells/CD62L^(high)CD25⁺cells in CD4⁺ T-cells is high due to cellinfusion at a stage where tumor regression has stopped (3.70→9.09), butthe ratio of (CD62L^(low) cells in CD4⁺ T-cells)/(CD62L^(high)CD25⁺cellsin CD4⁺ T-cells) is reduced (4.55) when tumor again turns to anincrease. This result shows that an effect of tumor regression isachieved by CD62L^(low) cells in a CD4⁺ T-cell population, not CD62Lhigh expression cells such as CD62L^(high)CD25⁺cells, and it ispreferable to remove CD62L high expression cells from a cell containingcomposition achieving an anti-tumor effect.

Example 5: Isolation/Expansion of CD62L^(low) Cells

CD62L staining patterns were observed for different races and mice.FIGS. 15A-15D show the results. Panel A (FIG. 15A) shows FACS using alymph node draining a tumor vaccine of a Caucasian. CD62L was observedwhile gating a lymphocyte region. FIG. 15C is a similar observation ofCD62L from peripheral blood derived mononuclear cells of Japanesesubjects. Panel D (FIG. 15D) shows CD62L staining patterns inlymphocytes of mice. It is understood that similar staining patterns areexhibited across human races/organism species. This has a double peakdistribution, with fluorescence intensity of 10² as the boundary.

Panel B (FIG. 15B) is FACS showing the purity after separating onlyCD62L^(low) cells from the group of cells of a subject in panel A withmagnetic beads. A cell population with fluorescence intensity exceeding10² was able to be nearly completely depleted. After separating thecells, pseudo-TCR stimulation was applied, and cells were cultured undera low concentration of IL-2. It was possible to expand the cell count1000-fold or more.

Example 6: Utilization of Marker Expressed on Dendritic Cells

6-1. Objective

The relationship between a therapeutic effect of an anti-PD-1 antibodyand a marker expressed on a dendritic cell was investigated. It wasexamined whether a marker expressed on dendritic cells can be utilizedin predicting a clinical effect of the cancer immunotherapy in thepresent invention.

6-2. Materials and Methods

Materials and methods are the same as Example 1. The antibodies shown inFIG. 23 were used to detect HLA-DR and CD80/CD86 expressed on dendriticcells. The approach to determination is the same as Example 1.

6-3. Results

FIG. 16 shows the results. The ratio of HLA-DR⁺cells and the ratio ofCD80 cells in myeloid dendritic cells (mDC, CD141⁺CD11c⁺ dendriticcells) and plasmacytoid dendritic cells (pDC, CD123⁺CD11c⁺ dendriticcells) were excellent indicators for identifying PD, SD, and PR. The pvalues when using HLA-DR in pDC, CD80 in pDC, HLA-DR in mDC, and CD80 inmDC for determining PD vs. PR+SD were 0.0008735, 0.002689351,6.87852×10⁻⁶, and 0.003033095, respectively, which are excellent values.As shown in FIG. 17, results of these markers in mDC were correlatedwith the ratio of CD62L^(low) CD4⁺ T-cells.

In view of the above results, the number/ratio of cells expressingHLA-DR and/or CD80 and/or CD86 in a myeloid dendritic cell (mDC) and/orplasmacytoid dendritic cell (pDC) population can be used as an indicatorinstead of (or in addition to) using CD4⁺ T-cells (CD62L^(low)CD4⁺T-cells) as an indicator.

Example 7: Utilization of Marker Expressed on CD8+ T-Cells

7-1. Objective

The relationship between a therapeutic effect of an anti-PD-1 antibodyand a marker expressed on a CD8+ T-cell was investigated. It wasexamined whether a marker expressed on CD8⁺ T-cells can be utilized inpredicting a clinical effect of cancer immunotherapy in the presentinvention.

7-2. Materials and Methods

Materials and methods are the same as Example 1. The antibodies shown inFIG. 23 were used to detect 4-1BB (CD137) expressed on CD8⁺ T-cells. Theapproach to determination is the same as Example 1.

7-3. Results

FIG. 18 shows the results. The ratio of HLA-DR⁺cells and the ratio ofCD80 cells in myeloid dendritic cells (mDC, CD141⁺CD11c⁺ dendriticcells) was correlated with the marker 4-1BB (CD137) expressed onCD62L^(low)CD8⁺ T-cells. As was in the results of Example 6, the resultof Example 7 shows that the number/ratio of 4-1BB cells in CD62L^(low)CD8⁺ T-cells can be utilized to predict the clinical effect of cancerimmunotherapy of the present invention in the same manner as thenumber/ratio of CD62L^(low)CD4⁺ T-cells.

Although not wishing to be bound by any theory, it is understood that(1) CD4⁺ T-cells transmits an instruction to dendritic cells via an MHCclass I molecule, thus increasing dendritic cells expressing HLA-DRand/or CD80 and/or CD86 and (2) the dendritic cells receiving theinstruction stimulate CD8⁺ T-cells via an MHC class II molecule, thusincreasing CD62L^(low) CD137(4-1BB)⁺CD8⁺ T-cells, so that thenumber/ratio of CD62L^(low) CD137(4-1BB)⁺CD8⁺ T-cells and dendriticcells expressing HLA-DR and/or CD80 and/or CD86 can be used inpredicting a clinical effect of cancer immunotherapy in the presentinvention in the same manner as the number/ratio of CD62L^(low)CD4⁺T-cells.

Furthermore, exhaustion in this series of anti-tumor mechanisms recoversdue to an anti-PD-1 antibody and an anti-PD-L1 antibody, while PD-1expression on T-cells is not effective for the prediction of the effectof the present invention (data not shown). In view of this result, it isunderstood that PD-L1 expression on dendritic cells can also be used inpredicting a clinical effect of cancer immunotherapy in the presentinvention.

Example 8: Utilization of Other Markers Expressed on CD4⁺ T-Cells

8-1. Objective

It was examined whether markers other than CD62L expressed on CD4⁺T-cells can be utilized in predicting a therapeutic effect.

8-2. Materials and Methods

Materials and methods are the same as Example 1. The antibodies shown inFIG. 23 were used to detect various markers expressed on CD4⁺ T-cells.The approach to determination is the same as Example 1.

8-3. Results

FIGS. 19A-19B and 20A-20D show the results. It is understood that eachof LAG3, ICOS, and CCR4 expressed on CD4⁺ T-cells can be used moreeffectively in predicting a clinical effect of cancer immunotherapy inthe present invention compared to CXCR3, CCR6, and CXCR5.

Example 9: Other Markers Separating PR and SD

9-1. Objective

Example 1 demonstrated that the percentage of CD25+Foxp3+CD4+ cells isan excellent marker for separating PR and SD. Other markers forseparating PR and SD were examined.

9-2. Materials and Methods

Materials and methods are the same as Example 1. The following markersusing an antibody for detecting ICOS expressed on CD4⁺ T-cells wereidentical to the antibodies used in Example 8. The approach todetermination is the same as Example 1.

9-3. Results

As is apparent from the results shown in FIG. 21, ICOS expressed on CD4⁺T-cells was found to be a better marker than Foxp3⁺CD25⁺. Furthermore,when the ratio of CD25⁺Foxp3⁺CD4⁺ T-cells in CD4⁺ T-cells (W) and theratio of ICOS⁺CD62L^(low)CD4⁺T-cells in CD62L^(low)CD4⁺ T-cells (Z) arecombined and used as the product Z*W for distinguishing a PR group froman SD group, this was found to be usable as a marker with sensitivity of80% and specificity of 89.5% when the threshold value of Z*W is 1.816(FIG. 21). This result also shows that PR and SD can be distinguishedusing a result of calculating (e.g., multiplying) two or more W of thepresent invention. One non-limiting Example can distinguish PR from SDby using variables (Z, W) such as Z*W or Z^(n)*W^(n) wherein n and m areeach positive real number, with an amount of an ICOS⁺CD62L^(low)CD4⁺T-cell subpopulation

as (Z) and a value selected from the group consisting of:an amount of a CD4⁺CD25⁺ T-cell subpopulation;an amount of a CD4⁺Foxp3⁺ T-cell subpopulation;an amount of a CD4⁺Foxp3⁺CD25⁺ T-cell subpopulation;an amount of a CD62L^(high)CD25⁺CD4⁺ T-cell subpopulation;an amount of a CD45RA⁻Foxp3⁺CD4⁺ T-cell subpopulation;an amount of a CCR4⁺CD25⁺CD4⁺ T-cell subpopulation; andan amount of a CD127⁺CD25⁺CD4⁺ T-cell subpopulation;as (W). It is also possible to use a result of calculating (e.g., addingand/or multiplying) three of more biomarkers for distinguishing PR fromSD.

To derive a more detailed formula for an indicator, logistic regressionwas performed to further examine a formula combining the ratio ofCD25⁺Foxp3⁺CD4⁺ T-cells in CD4⁺ T-cells (W) and the ratio ofICOS⁺CD62L^(low)CD4⁺ T-cells in CD62L^(low)CD4⁺ T-cells (Z).

As shown in FIGS. 24 and 25, the formula of J=Z*W⁵ was derived from theresult in a sample of N=32. When PR and SD were separated using theformula J=Z*W⁵ as an indicator, ROC analysis showed that the performancewas improved compared to each of Z and W (FIG. 26). In addition, it isunderstood that PR and SD can be successfully separated using anotherformula with a similar form, J=Z*W⁴⁻⁶.

Because CD4⁺ T cells are critical to predict PD-1/PD-L1 blockade therapyNR, the inventors next examined whether PD-1 and LAG-3 expression onCD4⁺ T cells can be a marker distinguishing PR group and SD group, inaddition to ICOS expression. The lymphocyte-activation gene 3 (LAG-3)protein, which is expressed on activated T cells, interacts with PD-1 tomaintain T cell exhaustion. LAG-3 binds to MHC class II antigens andregulates the expanding effector T cell population size followingantigen activation (28-30 Hui, E., et al. T cell costimulatory receptorCD28 is a primary target for PD-1-mediated inhibition. Science 355,1428-1433 (2017); Baixeras, E., et al. Characterization of thelymphocyte activation gene 3-encoded protein. A new ligand for humanleukocyte antigen class II antigens. J Exp Med 176, 327-337 (1992);Workman, C. J., et al. Lymphocyte activation gene-3 (CD223) regulatesthe size of the expanding T cell population following antigen activationin vivo. J Immunol 172, 5450-5455 (2004)). The inventors thus examinedPD-1, LAG-3, and ICOS expression on gated CD62L^(high) and CD62L^(low)CD4⁺ T cells.

The result is shown in FIGS. 32a-32f . These molecules were expressed onCD62L^(low) CD4⁺ T cells but minimally detected on CD62L^(high) CD4⁺ Tcells (FIG. 32d-e ). Post hoc tests following one-way analysis ofvariance (ANOVA) showed, notably, that IR (SD) possessed significantlylower PD-1⁺, LAG-3⁺′ and ICOS⁺cell percentages in the totalCD62L^(low)CD4⁺ T cell population compared with GR (PR) and NR (FIG.32a-c ). It is likely that IR had a CD4⁺ T cell immunity state distinctfrom that of GR. Thus, it is possible to distinguish PR from SD usingthe amount of these cell subpopulations.

It is understood from the result of this Example, that the amount/ratioof LAG-3⁺CD62L^(low) CD4⁺ T-cell and PD-1⁺CD62L^(low) CD4⁺ T-cellsubpopulation can be used as a marker to distinguish PR and SD, inaddition to ICOS⁺CD62L^(low) CD4⁺ T-cell.

Example 10: Survival Analysis

10-1. Summary

In order to demonstrate the prediction of therapeutic effect with theprediction formula (X²/Y, wherein X=the ratio of CD62L^(low) T-cells inthe CD4⁺ T-cell population (%) and Y=the ratio of CD25⁺FOXP3⁺ T-cells inthe CD4⁺ T-cell population (%)), the survival duration is analyzed inthe discovery cohort (a portion of subject patient population ofExample 1) whose treatment outcome (PD, SD, CR) have been determined.Further, in an independent validation cohort consisting of 41 patientscontinuing a treatment, whose prediction formulas were analyzed beforeassessment of tumor responsiveness, whether the prediction formula coulddistinguish NR (PD) was examined.

10-2. Materials and Methods

Characteristics of the patient group included in the discovery cohortand the validation cohort were as shown in the following table. Theprediction formula values for each patient were calculated in accordancewith the procedure described in Example 1.

TABLE 4 a. Patient characteristics Discovery cohort: n = 40 Validationcohort: n = 41 Age-yr Age-yr  Median 67  Median 71  Range 51-84  Range38-85 Sex-no. (%) Sex-no. (%)  Male 26 (65)    Male 35 (85.4)   Female14 (35)    Female 6 (14.6) Histology-no. (%) Histology-no. (%)  Sq 10(25)    Sq 10 (24.4)   Non-sq 30 (75)    Non-sq 31 (75.6)  Smokinghistory-no. (%) Smoking history-no. (%)  Current or former smoker 29(72.5)  Current or former smoker 38 (92.7)   Never smoked 11 (27.5) Never smoked 3 (7.3)  Disease stage-no. (%) Disease stage-no. (%) c-stageIII  9 (22.5)  c-stageIII 9 (22.0)  c-stageIV 22 (55)   c-stageIV 25 (61.0)   post-operative recurrence  9 (22.5) post-operative recurrence 7 (17.1) EGFR status-no. (%) EGFR status-no.(%)  Wild type 33 (82.5)  Wild type 41 (100)    Mutated (Exon19 del orL858R)  7 (17.5)  Mutated (Exon19 del or L858R) 0 (0)  

10-3. Results

The prediction formula (X²/Y, wherein X=the ratio of CD62L^(low) T-cellsin the CD4⁺ T-cell population (%) and Y=the ratio of CD25⁺FOXP3⁺ T-cellsin the CD4⁺ T-cell population (%)) values for each patient in thediscovery cohort are shown in FIG. 30a (P<0.0047, t=3.004, df=38). Theprediction formula receiver operating characteristic (ROC) analysis todetect NR at 8 weeks within the discovery cohort is shown in FIG. 30b .Sensitivity and specificity were 85.7% and 100%, respectively, at theprediction formula threshold value=192. The progression-free survival(PFS) and OS curves of patients diagnosed as responder type (X²/Y>=192)and NR type (X²/Y<192) according to PBMCs obtained before Nivolumabtreatment are shown in FIG. 30 c and d. Responder and NR types in thediscovery cohort (threshold=192) differed significantly (P<0.0001) inboth PFS and OS.

Next, the Inventors explored whether the prediction formula thresholdvalue (X²/Y<192) could differentiate NRs in the independent validationcohort consisting of 41 consecutive patients whose peripheral blood wascollected prior to Nivolumab therapy as the discovery cohort but wasanalyzed before tumor response evaluation. The prediction formula valueswere significantly higher (P=0.00068, t=3.693, df=39) in respondingvalidation cohort patients as shown in FIG. 30e . The sensitivity andspecificity values of NR validation cohort patient prediction were 90.9%and 89.5% at the <192 threshold, respectively. Responder-type PFS wassignificantly longer than NR-type in validation cohort patients (FIG.30f ; P<0.0001). Although the median follow-up time was only 195 days,responder-type patients also had significantly longer OS (FIG. 30g ;P=0.0022).

The objective responses at 8 week in each cohort were as follows.

TABLE 5 b. Responses to Nivolumab Discovery cohort: n = 40 Validationcohort: n = 41 Objective response at 8 weeks-no. (%) Objective responseat 8 weeks-no. (%) Complete or partial response 11 (27.5) Complete orpartial response  7 (17.1) Stable disease 15 (37.5) Stable disease 12(29.3) Progressive disease 14 (35) Progressive disease 22 (53.7)Abbreviations: Sq, squamous; c-stage, clinical stage; del, deletion.

The results in this Example showed that the method of predictingresponsiveness to cancer immunotherapy described herein predictsaccurately responsiveness to cancer immunotherapy, also in prospectivestudy. Further, prediction of responsiveness to cancer immunotherapyalso provides direct prediction of overall treatment response (overallsurvival (OS) or progression-free survival (PFS)) of patients.

Example 11: The Availability of CD28⁺Cells Subpopulation as a Marker

Recently, it was demonstrated that CD28, but not the T cell receptor(TCR), is a primary target of PD-1-dependent signal inhibition. Thus,the Inventors examined if the percentage of CD28⁺cells in the totalpopulation of CD8⁺ T cells correlated with the prediction formula value.

The Inventors found that the prediction formula (X²/Y, wherein X=theratio of CD62L^(low) T-cells in the CD4⁺ T-cell population (%) and Y=theratio of CD25⁺FOXP3⁺ T-cells in the CD4⁺ T-cell population (%)) valuessignificantly (P=0.0045) correlated with the percentage of CD28⁺cells inthe total population of CD62L^(low)CD8⁺ T cell (FIG. 31). From theresults of this Example, it is understood that an amount ofCD28⁺CD62L^(low)CD8⁺ T-cell and/or a ratio of CD28⁺cells in aCD62L^(low)CD8⁺ T-cell population can be used for prediction ofresponsiveness to cancer immunotherapy of patients, as well as saidprediction formula value.

Example 12: CD62Llow CD4+ T Cell Gene Expression in Patients of EachGroup

12-1. Summary

PBMC flow cytometry (FCM) analyses revealed that CD62L^(low)CD4⁺ T cellquantity and quality play a critical role in antitumor immunity anddetermine PD-1 blockade therapy response. The inventors performedmicroarray analysis to view CD62L^(low)CD4⁺ T cell differences at themolecular level among GR, IR, and NR patients in this Example. TheInventors first elucidated gene expression differences inCD62L^(high)CD4⁺and CD62L^(low) CD4⁺ T cells. Then the differentiallydifferential expressed gene on CD62L^(low) CD4⁺ T cells of the patientsof each group was explored.

12-2. Materials and Methods

Total RNAs were isolated by TRIzol reagent (Thermo Fisher Science,Waltham, Mass.) from CD62L^(high) CD4⁺and CD62L^(low) CD4⁺ T cells inPBMCs purified from two of each responder type. cDNA and cRNA synthesisand single-stranded cDNA (ssDNA) labeling reactions were performedaccording to the manufacture's instruction using the WT Plus Reagent Kit(Thermo Fisher Scientific). Total RNA (0.5 μg) was reverse transcribedinto cDNA and subsequently synthesized into cRNA. ssDNA was reversetranscribed from 15 μg of cRNA and then labeled; 1.8 μg of labeled ssDNAwas hybridized with microarray Clariom S assays for Human (Thermo FisherScientific) in a GeneChip Hybridization Oven 645. Hybridized arrays werescanned using the GCS3000 7G System (Thermo Fisher Scientific). Theaccession number ID of the gene expression data is GSE103157.

To identify the gene expression signature from two sets of geneexpression data, the Inventors estimated the difference of geneexpression between the two sets as follows. First, the Inventorsperformed the outlier test for all values of probes, and then calculateda z-score for each probe using the average and the variance of the probevalues except for outliers. To compare two gene sets of z-scores, thez-score of each gene was transformed into probability, and then eachdifference of gene probability between the two sets, p^(d) wascalculated; i.e.,

$\begin{matrix}{p_{k}^{d} = {{{{p\left( z_{k}^{a} \right)} - {p\left( z_{k}^{b} \right)}}} = {{{\frac{1}{\sqrt{2\pi}}{\int_{- \infty}^{z_{k}^{a}}{e^{- \frac{z}{2}}dz}}} - {\frac{1}{\sqrt{2\pi}}{\int_{- \infty}^{z_{k}^{b}}{e^{- \frac{z}{2}}{dz}}}}}}}} & \left\lbrack {{Math}.\mspace{14mu} 1} \right\rbrack\end{matrix}$

where the k-th gene between the two gene sets, a and b, was compared. Inthis analysis, the Inventors selected the genes with

p _(k) ^(d)>0.2  [Math. 2]

as the gene signature.

12-3. Results

For this, the Inventors first elucidated gene expression differences inCD62L^(high)CD4⁺and CD62L^(low) CD4⁺ T cells. CD62L^(high) CD4⁺ T cellsand CD62L^(low) CD4⁺ T cells have distinct gene expression profiles(FIGS. 33a and 34a ). Consistent with previous reports, the majority ofCD62L^(high) CD4⁺ T cells are considered as naive T cells because theC—C chemokine receptor type 7 (CCR7), CD28, and transcription factor 7(TCF7) genes were highly expressed in CD62L^(high) CD4⁺ T cells in allGR, IR, and NR patients. A few CD62L^(high) CD4⁺ T cells are consideredregulatory T cells because of higher foxp3 expression.

Then, the Inventors merged the genes in the signatures compared betweenthe cells from GR and IR, GR and NR, IR and NR, GR+IR and NR, and GR andIR+NR (1884, 1826, 1410, 1167, and 1513 genes, respectively) (totaling3484) (FIG. 33b ). Among these, the expression of 30 from 53 genes knownto be related to T-cell immunity was shown in terms ofNivolumab-treatment response (FIG. 34b ). This indicated that C-X-Cchemokine receptor type 3 (CXCR3), interleukin-23 receptor (IL23R),interleukin-13 receptor subunit alpha-2 (IL13RA2), PD-1 ligand 2 (PDL2),CD80, C-type lectin domain family 2 member A (CLEC2A), interleukin 7(IL7), transforming growth factor beta receptor 3 (TGFBR3), and histonedeacetylase 9 (HDAC9) were preferentially expressed in CD62L^(low) CD4⁺T cells derived from GR and/or IR.

As can be understood from the results of this Example, it is possible todetermine a cell subpopulation that an obtained cell belongs to byexamining the expression of differentially expressed genes betweenCD62L^(high)CD4⁺and CD62L^(low) CD4⁺ T-cells, and it is thus possible tomeasure an amount and/or ratio of a cell subpopulation. Further it isunderstood that distinction of patients group can be achieved byexamining the expression of differential expressed genes amongrespective patients groups on CD62L^(low) CD4⁺ T-cells.

Example 13: Cell Transfer Experiment

Preparation of CD62L^(low) CD4⁺ T cells from tumor-draining lymph nodes1.5×10⁶ B16BL6 melanoma cells (in HBSS) was inoculated to B6 micesubcutaneously. Inguinal lymph nodes were harvested 9 to 10 days after.From harvested lymph nodes, CD4⁺ T-cells were isolated with CD4⁺ T-cellIsolation Kit+LS column. CD62L^(low)CD4⁺ T-cells were purified bynegative selection with CD62L microbeads (LS column). TheseCD62L^(low)CD4⁺ T-cells were used for intravenous transfer.

Tumor Model

3×10⁶ B16BL6 melanoma cells in HBSS were inoculated on midline ofabdomen B6 mice subcutaneously. The mice were divide into (1) Controlgroup (N=10), (2) Antibody group (N=17), and (3) Antibody+Cell group(N=4). No treatment was administered to Control group. After inoculatingtumor cells to the Antibody+Cell group, the above CD62L^(low)CD4⁺T-cells (1*10⁶) were transferred on Day 4 or Day 5 after tumorinoculation, and anti-PD-1 antibody (RMP1-14 BioXcell 250m) wasadministered intraperitoneally (on the day of cell administration, 3days after and 6 days after cell administration). For the Antibodygroup, anti-PD-1 antibody (RMP1-14 BioXcell 250m) was administeredintraperitoneally (on the day of cell administration, 3 days after and 6days after cell administration). The survival ratio of mice in eachgroup was monitored.

Results

On day 16 after the administration of the antibody and/or T-cells, allindividuals in Control group died, while the survival ratio of theAntibody+Cell group was 50%, which was higher than that of the Antibodygroup (FIG. 35). These results showed that the transfer ofCD62L^(low)CD4⁺ T-cells can enhance the efficacy of anti PD-1antibodies.

INDUSTRIAL APPLICABILITY

Anti-PD-1/PD-L1 antibodies are considered as primary therapy for almostall progressive cancer therapy. Meanwhile, the Ministry of Health,Labour and Welfare has warned that expensive drug costs can potentiallyincrease the social security cost, so that the incrementalcost-effectiveness ratio (ratio of increase in therapeutic effect toincrease in drug cost) must be increased. The biomarkers provided in thepresent invention are medically and socially essential because they canpredict an effect of an anti-PD-1/PD-L1 antibody in a simple, low cost,and accurate manner. The present invention is understood as a techniquethat is demanded worldwide for all cancers and tumors, thus having avery high market value.

1. A method of predicting a response to cancer immunotherapy of asubject, the method comprising: measuring an amount of CD4⁺CD62L^(low)T-cells of the subject (X); comparing X with an ineffective groupthreshold value, thereby predicting that the subject is not a part of anineffective group to the cancer immunotherapy; and applying a cancerimmunotherapy to the subject predicted to be not a part of anineffective group; wherein the cancer immunotherapy comprisesadministration of an immune checkpoint inhibitor.
 2. The method of claim1, further comprising using a ratio of a Foxp3⁺CD25⁺ T-cellsubpopulation in CD4⁺ T-cells, a ratio of an ICOS⁺CD62L^(low)CD4⁺ T cellsubpopulation in CD62L^(low)CD4⁺ T-cells, a ratio ofLAG-3⁺CD62L^(low)CD4⁺ T cell subpopulation in CD62L^(low)CD4⁺ T-cells,or a ratio of PD-1⁺CD62L^(low)CD4⁺ T cell subpopulation inCD62L^(low)CD4⁺ T-cells, in the subject who is shown to be not a part ofthe ineffective group as an indicator of a response to the cancerimmunotherapy of the subject, wherein the ratio of the Foxp3⁺CD25⁺T-cell subpopulation in the CD4⁺ T-cells, the ratio of theICOS⁺CD62L^(low)CD4⁺ T cell subpopulation in the CD62L^(low)CD4⁺T-cells, the ratio of LAG-3⁺CD62L^(low)CD4⁺ T cell subpopulation inCD62L^(low)CD4⁺ T-cells, or the ratio of PD-1⁺CD62L^(low)CD4⁺ T cellsubpopulation in CD62L^(low)CD4⁺ T-cells, higher than a response groupthreshold value indicates that the subject is a part of a responsegroup.
 3. The method of claim 1, wherein the ineffective group thresholdvalue is determined so that sensitivity for the detection of anineffective group exceeds about 90%, or wherein the ineffective groupthreshold value is determined so that specificity for the detection ofan ineffective group exceeds about 90%.
 4. The method of claim 1,wherein the amount of the cell population is measured using a peripheralblood sample.
 5. The method of claim 1, wherein the immune checkpointinhibitor is selected from the group consisting of a PD-1 inhibitor anda PD-L1 inhibitor.
 6. The method of claim 5, wherein the PD-1 inhibitoror PD-L1 inhibitor comprises nivolumab, pembrolizumab, durvalumab,atezolizumab, or avelumab.
 7. The method of claim 1, wherein the X is aratio of a CD62L^(low)CD4⁺ T-cell subpopulation in CD4⁺ T-cells.